A Comprehensive Review of Genetically Engineered Mouse Models for Prader-Willi Syndrome Research

Prader-Willi syndrome (PWS) is a neurogenetic multifactorial disorder caused by the deletion or inactivation of paternally imprinted genes on human chromosome 15q11-q13. The affected homologous locus is on mouse chromosome 7C. The positional conservation and organization of genes including the imprinting pattern between mice and men implies similar physiological functions of this locus. Therefore, considerable efforts to recreate the pathogenesis of PWS have been accomplished in mouse models. We provide a summary of different mouse models that were generated for the analysis of PWS and discuss their impact on our current understanding of corresponding genes, their putative functions and the pathogenesis of PWS. Murine models of PWS unveiled the contribution of each affected gene to this multi-facetted disease, and also enabled the establishment of the minimal critical genomic region (PWScr) responsible for core symptoms, highlighting the importance of non-protein coding genes in the PWS locus. Although the underlying disease-causing mechanisms of PWS remain widely unresolved and existing mouse models do not fully capture the entire spectrum of the human PWS disorder, continuous improvements of genetically engineered mouse models have proven to be very powerful and valuable tools in PWS research.

Transferability of apple and pear SSRs to other temperate pome fruit crops of family Rosaceae

Extensive use of simple sequence repeat (SSR) is facilitated if loci would be transferable across species even in closely related genera to overcome high cost and efforts involved in their development as major constraints. In the present study, apple and pear genomic microsatellite primer pairs were used to amplify SSR loci in apple, pear, quince and loquat genotypes, respectively. Already reported SSRs were selected based on their polymorphic survey for successful amplification with at least one polymerase chain reaction (PCR) product of the approximate size expected for a homologous locus screened among apple and pear genotypes for further transferability exploration across other temperate pome fruit crops, respectively. Highest transferability of apple and pear SSR, 61.53 % and 73.33 % was observed in closely related quince and apple genotypes, respectively. This indicated that primer binding sites between these two closely related genera, Malus and Pyrus, are fairly well conserved. Maximum transferability rate was found to be 93.33 % and 80.00 % across all the subjected genotypes for primer CH05D11 and TSUenh016 in apple and pear, respectively. The transferability of markers is based on genomic similarity, and can reflect the relationship of genome collinearity and even evolution between species. This high level of transferability of apple and pear SSRs to other temperate pome fruit crops indicated their promise for application to future molecular screening, map construction, and comparative genomic studies, etc.

Comparative analysis of the complete chloroplast genome sequences of three Amaranthus species

The complete chloroplast (cp) genome sequences of three Amaranthus species (Amaranthus hypochondriacus, A. cruentus and A. caudatus) were determined by next-generation sequencing. The cp genome sequences of A. hypochondriacus, A. cruentus and A. caudatus were 150,523, 150,757 and 150,523 bp in length, respectively, each containing 84 genes with identical contents and orders. Expansion or contraction of the inverted repeat region was not observed among the three Amaranthus species. The coding regions were highly conserved with 99.3% homology in nucleotide and amino acid sequences. Five genes – matK, accD, ndhJ, ccsA and ndhF – showed relatively high non-synonymous/synonymous values (Ka/Ks > 0.1). Sequence comparison identified two insertion/deletion (InDels) greater than 40 bp in length, and polymerase chain reaction markers that could amplify these InDel regions were applied to diverse Korean Genbank accessions, which could discriminate the three Amaranthus species. Phylogenetic analyses based on 62 protein-coding genes showed that the core Caryophyllales were monophyletic and Amaranthoideae formed a sister group with the Betoideae and Chenopodioideae clade. Comparing each homologous locus among the three Amaranthus species, identified eight regions with high Pi values (>0.03). Seven of these loci, except for rps19-trnH (GUG), were considered to be useful molecular markers for further phylogenetic studies.

Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation ofBacillus subtilis

ABSTRACTDuring competence,Bacillus subtilisis able to take up DNA from its environment through the process of transformation. We investigated the ability ofB. subtilisto take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60 min is expressed on average 6 h 45 min after the addition of DNA and 4 h 45 min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCEWe used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process ofBacillus subtilisat the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species.

pSEUDO, a Genetic Integration Standard for Lactococcus lactis

ABSTRACTPlasmid pSEUDO and derivatives were used to show thatllmg_pseudo_10inLactococcus lactisMG1363 and its homologous locus inL. lactisIL1403 are suitable for chromosomal integrations.L. lactisMG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enablingin vivononinvasive monitoring of cellular fitness were constructed.

Differential Azole Antifungal Efficacies Contrasted Using a Saccharomyces cerevisiae Strain Humanized for Sterol 14α-Demethylase at the Homologous Locus

ABSTRACT Inhibition of sterol-14α-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14α-methyl-ergosta-8, 24(28)-dien-3β-6α-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.

Patterns of genome duplication within the Brassica napus genome

The progenitor diploid genomes (A and C) of the amphidiploid Brassica napus are extensively duplicated with 73% of genomic clones detecting two or more duplicate sequences within each of the diploid genomes. This comprehensive duplication of loci is to be expected in a species that has evolved through a polyploid ancestor. The majority of the duplicate loci within each of the diploid genomes were found in distinct linkage groups as collinear blocks of linked loci, some of which had undergone a variety of rearrangements subsequent to duplication, including inversions and translocations. A number of identical rearrangements were observed in the two diploid genomes, suggesting they had occurred before the divergence of the two species. A number of linkage groups displayed an organization consistent with centric fusion and (or) fission, suggesting this mechanism may have played a role in the evolution of Brassica genomes. For almost every genetically mapped locus detected in the A genome a homologous locus was found in the C genome; the collinear arrangement of these homologous markers allowed the primary regions of homoeology between the two genomes to be identified. At least 16 gross chromosomal rearrangements differentiated the two diploid genomes during their divergence from a common ancestor.Key words: genome evolution, Brassicaeae, polyploidy, homoeologous linkage groups.

Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

ABSTRACT Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGHoperon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgHgene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.