Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.Complexities of natural biological systems make it difficult to understand and define precisely the roles of individual genes and their integrated functions. The use of model organisms with a relatively small number of genes enables the isolation of core biological processes from their complex regulatory networks for extensive characterization. However, even the simplest natural microbes contain many genes of unknown function, as well as genes that can be singly or simultaneously deleted without any noticeable effect on growth rate in a laboratory setting (Hutchison et al. 1999; Glass et al. 2006; Posfai et al. 2006). Ill-defined genes and those mediating functional redundancies both compound the challenge of understanding even the simplest life forms.Toward generating a minimal cell where every gene is essential for the axenic viability of the organism, we are pursuing strategies to reduce the 1-Mb genome of Mycoplasma mycoides JCVI-syn1.0 (Gibson et al. 2010). Because we can (1) introduce this genome into yeast and maintain it as a plasmid (Benders et al. 2010; Karas et al. 2013a); and (2) “transplant” the genome from yeast into mycoplasma recipient cells (Lartigue et al. 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering. Here we further exploit distinctive features of yeast for this purpose.Methods for serially replacing genomic regions with selectable markers are limited by the number of available markers. One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al. 2001). We have previously used a self-excising marker (Noskov et al. 2010) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 ∆1-6) (Karas et al. 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming. When applied to poorly characterized genes with the potential to interact with other genes, some paths for multigene knockout may lead to dead ends that result from synergistic mutant phenotypes. When a dead end is reached, sequentially returning to a previous genome in an effort to find a detour to a viable higher-order multimutant may be prohibitively time-consuming.An alternative approach to multigene engineering, available in yeast, is to prepare a set of single mutants and combine the deletions into a single strain via cycles of mating and meiotic recombination (Fig. 1A; Pinel et al. 2011; Suzuki et al. 2011, 2012). With a green fluorescent protein (GFP) reporter gene inserted in each deletion locus, the enrichment of higher-order yeast deletion strains in the meiotic population can be accomplished using flow cytometry. Here we apply this method to the JCVI-syn1.0 ∆1-6 exogenous, bacterial genome harbored in yeast to nonsequentially assemble deletions for genes predicted to be individually deletable based on biological knowledge or transposon-mediated disruption data. The functional identification of simultaneously deletable regions is expected to accelerate the effort to construct a minimal genome.Open in a separate windowFigure 1.Progressive clustering of deleted genomic segments. (A) Scheme of the method. Light blue oval represents a bacterial cell. Black ring or horizontal line denotes a bacterial genome, with the orange box indicating the yeast vector used as a site for linearization and recircularization. Gray shape denotes a yeast cell. Green dot in the genome indicates a deletion replaced with a GFP marker. (B) Map of deleted regions. Orange box indicates the yeast vector sequence used for genome linearization and recircularization. Green boxes indicate regions deleted in multimutant mycoplasma strains. Blue boxes denote restriction modification (RM) systems that are also deleted in the strains. (C) Pulsed-gel electrophoresis result for deleted genomes. The starting strain was the JCVI-syn1.0 ∆1–6 strain (1062 kb). Two strains were analyzed for each design of simultaneous deletion (962 kb for eight-deletion or 974 kb for seven-deletion genome). Ladder is a set of yeast chromosomes (New England BioLabs). (D) GFP-RFP ratio sorting result. Standard sorting was compared with sorting based on a GFP-RFP ratio (Methods).     

Challenging issues in rheumatology: thoughts and perspectives

Clinical Rheumatology -     

Identification of an oncogenic form of the thrombopoietin receptor MPL using retrovirus-mediated gene transfer

Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3- dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.     

Visual Acuity and Retinal Changes in South Australian Aborigines

Summary: Visual acuity and retinal changes in South Australian Aborigines. F. M. Edwards, P. H. Wise, R. J. Craig, D. W. Thomas and J. B. Murchland
Eye examinations were carried out at four Aboriginal reserves. Of 361 Aboriginal adults tested, 64 had a visual defect (visual acuity of 6/9 or worse) in each eye, a prevalence of 18%, with an additional 79 (22%) with a similar loss of acuity in one eye only; these were more frequently seen at the urbanized reserve of Koonibba.
Only one full blood Aboriginal child within the less urbanized communities had a reduced visual acuity, whereas seven (10%) part blood children at a more urbanized reserve had reduced vision in both eyes, with a further nine (13%) in one eye only, not unlike figures quoted for South Australian school children.
Vascular changes in the fundus oculi were observed and occurred more often when hypertension and/or hyperglycaemia were present. They consisted of arteriovenous crossing changes (26%), swelling of the retinal veins (3%) and altered light reflex (41%) and were not infrequently seen in the younger adult (20% of adults under 30 years had AV crossing changes and 46% had widened light streak). Analysis suggests that, although hypertension and hyperglycaemia are related to retinal vascular changes, other factors, as yet unidentified, are present in the Aboriginal population under consideration.     

Is central nervous system processing altered in patients with heart failure?

AIMS: Breathlessness is a cardinal symptom of heart failure and the altered regulation of breathing is common. The contribution of abnormal central nervous system activity has not previously been investigated directly, although abnormal autonomic responses have been described. Our aim was to assess whether heart failure patients exhibit different patterns of regional brain activation after exercise stress. METHODS: We used positron emission tomography with H2(15)O, to measure changes in regional cerebral blood flow (rCBF) and absolute global cerebral blood flow (gCBF) in 6 male class II/III heart failure patients and 6 normal controls. Breathlessness (0-5 visual analogue scale) and respiratory parameters were measured at rest, after horizontal bicycle exercise and during isocapnic hyperventilation. CBF was measured in each condition in all subjects. RESULTS: Both groups were similarly breathless after exercise and the respiratory parameters were comparable. rCBF differences for the main comparison (exercise vs hyperventilation) were: activation of the right frontal medial gyrus (P < 0.001, Z = 4.90) and left precentral gyrus (P < 0.03, Z = 4.66) in controls but not in patients. Both groups had rCBF increases in the left anterior cingulate (P < 0.05, Z = 4.67) and right dorsal cingulate cortex (P < 0.05, Z = 4.66). The gCBF did not differ between exercise, isocapnic hyperventilation and rest in patients but, in controls, gCBF was greater after exercise compared to either isocapnic hyperventilation or rest. CONCLUSION: Heart failure patients had a distinct pattern of regional cortical activity with exercise-induced breathlessness but unvarying CBF values between conditions. These central neural differences in activity may contribute to some features of heart failure, such as variability in symptoms and autonomic dysregulation.     

Serial lung function testing in patients treated with amiodarone: a prospective study

PURPOSE: Amiodarone has proven to be effective in many cases of cardiac arrhythmias, refractory ventricular tachycardia, and ventricular fibrillation. Pulmonary toxicity is a possible side effect of the drug, with a reported incidence of 2 to 15 percent per year. To determine the effect of amiodarone on lung function, we prospectively studied serial lung function tests in a cohort of 91 patients with refractory cardiac arrhythmias treated with this agent. PATIENTS AND METHODS: Spirometry and carbon monoxide diffusing capacity (DLCO) were measured at zero, three, six, 12, 18, and 24 months, with a mean follow-up of 351 days. RESULTS: For the whole population taking a mean dose of amiodarone of 367 mg daily (range: 136 to 512 mg), there was no accelerated rate of decline in spirometric indices or DLCO. Analysis of lung function changes by multivariate analysis demonstrated that an accelerated decline in DLCO values occurred in elderly patients (p less than 0.05) but not in patients with pre-existing lung disease or cigarette smokers. In four patients (4.5 percent), clinical evidence of amiodarone pulmonary toxicity developed that was associated with a fall in DLCO of greater than 20 percent. All four patients recovered after the drug was stopped. Another 15 patients, without clinical evidence of pulmonary toxicity, had a sustained decline in DLCO of greater than 20 percent. These 15 patients remained asymptomatic over the next 11 months without interruption of therapy. A greater than 20 percent fall in DLCO was a sensitive test for clinically evident amiodarone pulmonary toxicity, but had a positive predictive value of only 21 percent. CONCLUSION: An isolated fall in DLCO, in the absence of clinical evidence of toxicity, does not necessitate stopping amiodarone. An unchanged DLCO value appears to be a reliable negative predictor of pulmonary toxicity.     

Comparison of biplane and single plane left ventricular volumes in atrial septal defect

The close agreement between biplane (BP) and single-plane (SP) angiographic estimates of left ventricular (LV) volumes results from the similarity of the minor axes measured in the right anterior oblique (RAO) and left anterior oblique (LAO) views. Disease states that alter LV geometry may change the length of one minor axis more than the other, producing a discrepancy between BP and SP volumes. To examine this hyposthesis, angiographically derived volumes in 21 patients with atrial septal defects (ASD) in which the LV appears to be compressed and flattened by an enlarged right ventricle, were compared to 100 normal control patients. In the control patients, the median SP estimate of end-diastolic volume (EDV) was 7.6% larger than the BP determination, whereas in patients with ASD, the median SP EDV estimate was 16.7% larger than the BP EDV (P<0.0001). The SP end-systolic volume (ESV) underestimated the BP value by 3.4% in controls but overestimated the BP ESV in patients with ASD by 4.3% (P<0.02). The overestimate of the SP EDV and SP ESV when compared to the BP volumes may be due to changes in either the minor axes or the appearance of the longest major axis in the LAO view. The longest major axis was found in the RAO view in 99% (99/100) of normals and 95% (20/21) of ASD patients (P?NS). The median ratio of RAO to LAO end-diastolic minor axes, however, was 1.07 in the normals and 1.17 for ASD patients. The median ratio of end-systolic minor axes was 0.97 for controls and 1.04 for ASD patients. Compression of the LV in patients with ASD shortens the LAO minor axis, resulting in a significantly greater SP overestimation of LV volume than occurs in normals. The degree of SP volume overestimate was not predicted by the magnitude of the left-to-right shunt or pulmonary pressure. This source of error affects all SP methods for determining left ventricular volume, including radionuclide techniques using static images.     

The association between sleep spindles and IQ in healthy school-age children

Recent studies have suggested that sleep is associated with IQ measures in children, but the underlying mechanism remains unknown. An association between sleep spindles and IQ has been found in adults, but only two previous studies have explored this topic in children. The goal of this study was to examine whether sleep spindle frequency, amplitude, duration and/or density were associated with performance on the perceptual reasoning, verbal comprehension, working memory, and processing speed subscales of the Wechsler Intelligence Scale for Children-IV (WISC-IV). We recruited 29 typically developing children 7–11 years of age. We used portable polysomnography to document sleep architecture in the natural home environment and evaluated IQ. We found that lower sleep spindle frequency was associated with better performance on the perceptual reasoning and working memory WISC-IV scales, but that sleep spindle amplitude, duration and density were not associated with performance on the IQ test.     

Genetic ancestries in northwest Cambodia

A survey of the genetic ancestry of 125 Cambodian children resident in Siem Reap province was undertaken, based on eight Y-chromosome binary polymorphisms and sequencing of the mtDNA HV1 region. The data indicated a largely East Asian paternal ancestry and a local Southeast Asian maternal ancestry. The presence of Y-chromosomes P* and R1a1* was suggestive of a small but significant Indo-European male ancestral component, which probably reflects the history of Indian, and later European, influences on Cambodia.     

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.