Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Unbiased prediction of off-target sites in genome-edited rice using SITE-Seq analysis on a web-based platform.

Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.

Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genes cooperatively with Hmo1 in Saccharomyces cerevisiae.

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.

Orthologs of Saccharomyces cerevisiae SFH2, genes encoding Sec14 family proteins, implicated in utilization of n-alkanes and filamentous growth in response to n-alkanes in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has an ability to assimilate n-alkanes as carbon and energy sources. In this study, the roles of orthologs of Saccharomyces cerevisiae SEC14 family gene SFH2, which we named SFH21, SFH22, SFH23 and SFH24, of Y. lipolytica were investigated. The transcript levels of SFH21, SFH22 and SFH23, determined by RNA-seq analysis, qRT-PCR analysis and northern blot analysis, were found to increase in the presence of n-alkanes. The deletion mutant of SFH21, but not that of SFH22, SFH23 or SFH24, showed defects in growth in the media containing n-alkanes and in filamentous growth on the solid media containing n-alkanes. Additional deletions of SFH22 and SFH23 significantly exaggerated the defect in filamentous growth of the deletion mutant of SFH21, and expression of SFH22 or SFH24 using the SFH21 promoter partially suppressed the growth defect of the deletion mutant of SFH21 on n-alkanes. These results suggest that SFH2 orthologs are involved in the utilization of n-alkanes and filamentous growth in response to n-alkanes in Y. lipolytica.

Identification and structural characterisation of a catecholate-type siderophore produced by Stenotrophomonas maltophilia K279a.

Siderophores are produced by several bacteria that utilise iron in various environments. Elucidating the structure of a specific siderophore may have valuable applications in drug development. Stenotrophomonas maltophilia, a Gram-negative bacterium that inhabits a wide range of environments and can cause pneumonia, produces siderophores. However, the structure was unknown, and therefore, in this study, we aimed to elucidate it. We purified siderophores from cultures of S. maltophilia K279a using preparative reversed-phase HPLC. The structure was analysed through LC-MS and 1H and 13C NMR. The results demonstrated that S. maltophilia K279a produces 2,3-dihydroxybenzoylserine (DHBS), a monomer unit of enterobactin. We suggested the uptake of Iron(III) by the DHBS complex. DHBS production by S. maltophilia K279a could be attributed to an incomplete enterobactin pathway. Drugs targeting DHBS synthesis could prevent S. maltophilia infection.

Draft genome sequencing of Sporolactobacillus terrae SBT-1, an efficient bacterium to ferment concentrated sugar to D-lactic acid.

Recently, the industrial-scale development of microbial D-lactic acid production has been discussed. In this study, the efficiency of the new isolate Sporolactobacillus terrae SBT-1 for producing D-lactic acid under challenge conditions was investigated. The isolate SBT-1 exhibited superior activity in fermenting a very high glucose or sucrose concentration to D-lactic acid compared to the other S. terrae isolates previously reported in the literature; therefore, SBT-1 could overcome the limitations of effective lactic acid production. In batch cultivation using 360 g/L glucose, SBT-1 produced 290.30 g/L D-lactate with a sufficiently high glucose conversion yield of 86%, volumetric productivity of 3.02 g/L h, and optical purity of 96.80% enantiomer excess. SBT-1 could also effectively utilize 440 g/L sucrose as a sole carbon source to produce 276.50 g/L lactic acid with a conversion yield of 90%, a production rate of 2.88 g/L h, and an optical purity of 98%. D-Lactic acid fermentation by two other related producers, S. inulinus NRIC1133T and S. terrae NRIC0357T, was compared with fermentation by isolate SBT-1. The experimental data revealed that SBT-1 possessed the ability to ferment relatively high glucose or sucrose concentrations to D-lactic acid without obvious catabolite repression and byproduct formation compared to the two reference strains. In draft genome sequencing of S. terrae SBT-1, the results provided here can promote further study to overcome the current limitations for the industrial-scale production of D-lactic acid.

Comparative Genomic Analysis of Closely Related Acetobacter pasteurianus Strains Provides Evidence of Horizontal Gene Transfer and Reveals Factors Necessary for Thermotolerance.

Acetobacter pasteurianus is an industrial strain used for the vinegar production. Many A. pasteurianus strains with different phenotypic characteristics have been isolated so far. To understand the genetic background underpinning these phenotypes, a comparative genomic analysis of A. pasteurianus strains was conducted. Based on bioinformatics and experimental results, we report the following. (i) The gene repertoire related to the respiratory chains showed that several horizontal gene transfer events occurred after the divergence of these strains, indicating that the respiratory chain in A. pasteurianus has the diversity to adapt to its environment. (ii) There is a clear difference in thermotolerance even between 12 closely related strains. NBRC 3279, NBRC 3284, and NBRC 3283, in particular, which have only 55 mutations in total, showed differences in thermotolerance. The Na+/H+ antiporter gene nhaK2 was mutated in the thermosensitive NBRC 3279 and NBRC 3284 strains and not in the thermotolerant NBRC 3283 strain. The Na+/H+ antiporter activity of the three strains and expression of nhaK2 gene from NBRC 3283 in the two thermosensitive strains showed that these mutations are critical for thermotolerance. These results suggested that horizontal gene transfer events and several mutations have affected the phenotypes of these closely related strains.IMPORTANCEAcetobacter pasteurianus, an industrial vinegar-producing strain, exhibits diverse phenotypic differences such as respiratory activity related to acetic acid production, acetic acid resistance, or thermotolerance. In this study, we investigated the correlations between genome sequences and phenotypes among closely related A. pasteurianus strains. The gene repertoire related to the respiratory chains showed that the respiratory components of A. pasteurianus has a diversity caused by several horizontal gene transfers and mutations. In three closely related strains with clear differences in their thermotolerances, we found that the insertion or deletion that occurred in the Na+/H+ antiporter gene nhaK2 is directly related to their thermotolerance. Our study suggests that a relatively quick mutation has occurred in the closely related A. pasteurianus due to its genetic instability and that this has largely affected its phenotype.

PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces.

BACKGROUND: Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. RESULTS: We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. CONCLUSIONS: These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Rhizobium dioscoreae sp. nov., a plant growth-promoting bacterium isolated from yam (Dioscorea species).

This study investigated endophytic nitrogen-fixing bacteria isolated from two species of yam (water yam, Dioscorea alata L.; lesser yam, Dioscorea esculenta L.) grown in nutrient-poor alkaline soil conditions on Miyako Island, Okinawa, Japan. Two bacterial strains of the genus Rhizobium, S-93T and S-62, were isolated. The phylogenetic tree, based on the almost-complete 16S rRNA gene sequences (1476 bp for each strain), placed them in a distinct clade, with Rhizobium miluonense CCBAU 41251T, Rhizobium hainanense I66T, Rhizobium multihospitium HAMBI 2975T, Rhizobium freirei PRF 81T and Rhizobium tropici CIAT 899T being their closest species. Their bacterial fatty acid profile, with major components of C19 : 0 cyclo ω8c and summed feature 8, as well as other phenotypic characteristics and DNA G+C content (59.65 mol%) indicated that the novel strains belong to the genus Rhizobium. Pairwise average nucleotide identity analyses separated the novel strains from their most closely related species with similarity values of 90.5, 88.9, 88.5, 84.5 and 84.4 % for R. multihospitium HAMBI 2975T, R. tropici CIAT 899T, R. hainanense CCBAU 57015T, R. miluonense HAMBI 2971T and R. freirei PRF 81T, respectively; digital DNA-DNA hybridization values were in the range of 26-42 %. Considering the phenotypic characteristics as well as the genomic data, it is suggested that strains S-93T and S-62 represent a new species, for which the name Rhizobium dioscoreae is proposed. The type strain is S-93T (=NRIC 0988T=NBRC 114257T=DSM 110498T).

Enterococcus florum sp. nov., isolated from a cotton flower (Gossypium hirsutum L.).

A Gram-stain-positive and catalase negative coccus, designated strain Gos25-1T, isolated from a cotton flower (Gossypium hirsutum L.) collected from Khao Wong district, Kalasin province, Thailand. The taxonomic position of this strain was systematically studied based upon polyphasic taxonomic methods. The strain was facultatively anaerobic and produced l-lactic acid from glucose. The predominant cellular fatty acids were the straight-chain fatty acids C18 : 1ω9c and C16 : 0. According to 16S rRNA and phenylalanyl-tRNA synthase alpha subunit (pheS) gene sequence similarity, this strain was closely related to Enterococcus pallens NBRC 100697T, E. hermanniensis CIP 108559T, E. avium NBRC 100477T and E. raffinosus NBRC 100492T with 98.9-99.1 % and 77.0-82.0 % sequence similarities, respectively. Phylogenetic analysis indicated that strain Gos25-1T was clearly distinguished from closely related species of the genus Enterococcus. Draft genome of Gos25-1T had a size of 3.99 Mb which was contained 3788 coding sequences with in silico G+C content of 42.4 mol%. The ANIb and a digital DNA-DNA hybridisation (dDDH) values between strain Gos25-1T and the closest related species, E. pallens NBRC 100697T were 73.65 and 21.10 %, respectively. According to polyphasic characterisation, this strain represents a novel species of the genus Enterococcus, for which the name Enterococcus florum sp. nov. is proposed. The type strain is Gos25-1T (=CIP 110956T=LMG 29007T=NBRC 111461T=TISTR 2382T).

A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloisomaltooligosaccharides.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-D-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-D-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Essentiality of WalRK for growth in Bacillus subtilis and its role during heat stress.

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

Genetic Predisposition to Ischemic Stroke: A Polygenic Risk Score.

BACKGROUND AND PURPOSE: The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk. METHODS: We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets). RESULTS: In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33-2.31) and 1.99 (95% confidence interval, 1.19-3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001). CONCLUSIONS: The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors.

Population Evolution of Helicobacter pylori through Diversification in DNA Methylation and Interstrain Sequence Homogenization.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

Δ12-fatty acid desaturase is involved in growth at low temperature in yeast Yarrowia lipolytica.

We investigated the role of FAD2, which was predicted to encode a fatty acid desaturase of the n-alkane-assimilating yeast Yarrowia lipolytica. Northern blot analysis suggested that FAD2 transcription was upregulated at low temperature or in the presence of n-alkanes or oleic acid. The FAD2 deletion mutant grew as well as the wild-type strain on glucose, n-alkanes, or oleic acid at 30 °C, but grew at a slower rate at 12 °C, when compared to the wild-type strain. The growth of the FAD2 deletion mutant at 12 °C was restored by the addition of 18:2, but not 18:1, fatty acids. The amount of 18:2 fatty acid in the wild-type strain was increased by the incubation at 12 °C and in the presence of n-octadecane. In contrast, 18:2 fatty acid was not detected in the deletion mutant of FAD2, confirming that FAD2 encodes the Δ12-fatty acid desaturase. These results suggest that Δ12-fatty acid desaturase is involved in the growth of Y. lipolytica at low temperature.

Distribution of human single-nucleotide polymorphisms is approximated by the power law and represents a fractal structure.

Single-nucleotide polymorphisms (SNPs) are one of the main causes of evolution. The distribution of human SNPs, which were examined in detail genomewide, was analyzed. Three discrete databases of human SNPs were used for this analysis, and similar results were obtained from these databases. It was found that the distribution of the distance between SNPs was approximated by the power law, and the shape of the regions including SNPs had the so-called fractal structure. Although the reason why the distribution of SNPs obeys such a certain law of physics is unclear, a speculation was attempted in connection with the three-dimensional structure of human chromatin which has a fractal structure.

The tap-tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei.

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.