Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have ...been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the
ltrB gene of
Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of
Clostridium acetobutylicum and 5 of
Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in
Clostridium botulinum and
Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
Pitt-Hopkins syndrome (PHS) is a rare syndromic encephalopathy characterized by daily bouts of hyperventilation and a facial gestalt. We report a 1.8-Mb de novo microdeletion on chromosome 18q21.1, ...identified by array–comparative genomic hybridization in one patient with PHS. We subsequently identified two de novo heterozygous missense mutations of a conserved amino acid in the basic region of the
TCF4 gene in three additional subjects with PHS. These findings demonstrate that
TCF4 anomalies are responsible for PHS and provide the first evidence of a human disorder related to class I basic helix-loop-helix transcription-factor defects (also known as “E proteins”). Moreover, our data may shed new light on the normal processes underlying autonomic nervous system development and maintenance of an appropriate ventilatory neuronal circuitry.
By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (
NFIX) deletions or nonsense mutation in three sporadic cases of a ...Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome,
Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider
NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice
NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that
NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic
NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.
Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres-the ends of ...linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.
We generated high-resolution maps of histone H3 lysine 9/14 acetylation (H3ac), histone H4 lysine 5/8/12/16 acetylation (H4ac), and histone H3 at lysine 4 mono-, di-, and trimethylation (H3K4me1, ...H3K4me2, H3K4me3, respectively) across the ENCODE regions. Studying each modification in five human cell lines including the ENCODE Consortium common cell lines GM06990 (lymphoblastoid) and HeLa-S3, as well as K562, HFL-1, and MOLT4, we identified clear patterns of histone modification profiles with respect to genomic features. H3K4me3, H3K4me2, and H3ac modifications are tightly associated with the transcriptional start sites (TSSs) of genes, while H3K4me1 and H4ac have more widespread distributions. TSSs reveal characteristic patterns of both types of modification present and the position relative to TSSs. These patterns differ between active and inactive genes and in particular the state of H3K4me3 and H3ac modifications is highly predictive of gene activity. Away from TSSs, modification sites are enriched in H3K4me1 and relatively depleted in H3K4me3 and H3ac. Comparison between cell lines identified differences in the histone modification profiles associated with transcriptional differences between the cell lines. These results provide an overview of the functional relationship among histone modifications and gene expression in human cells.
We present an analysis of chromatin fiber structure across the human genome. Compact and open chromatin fiber structures were separated by sucrose sedimentation and their distributions analyzed by ...hybridization to metaphase chromosomes and genomic microarrays. We show that compact chromatin fibers originate from some sites of heterochromatin (C-bands), and G-bands (euchromatin). Open chromatin fibers correlate with regions of highest gene density, but not with gene expression since inactive genes can be in domains of open chromatin, and active genes in regions of low gene density can be embedded in compact chromatin fibers. Moreover, we show that chromatin fiber structure impacts on further levels of chromatin condensation. Regions of open chromatin fibers are cytologically decondensed and have a distinctive nuclear organization. We suggest that domains of open chromatin may create an environment that facilitates transcriptional activation and could provide an evolutionary constraint to maintain clusters of genes together along chromosomes.
Copy number variants (CNVs) underlie many aspects of human phenotypic diversity and provide the raw material for gene duplication and gene family expansion. However, our understanding of their ...evolutionary significance remains limited. We performed comparative genomic hybridization on a single human microarray platform to identify CNVs among the genomes of 30 humans and 30 chimpanzees as well as fixed copy number differences between species. We found that human and chimpanzee CNVs occur in orthologous genomic regions far more often than expected by chance and are strongly associated with the presence of highly homologous intrachromosomal segmental duplications. By adapting population genetic analyses for use with copy number data, we identified functional categories of genes that have likely evolved under purifying or positive selection for copy number changes. In particular, duplications and deletions of genes with inflammatory response and cell proliferation functions may have been fixed by positive selection and involved in the adaptive phenotypic differentiation of humans and chimpanzees.
In many countries relatively high notification rates of campylobacteriosis are observed in children under 5 years of age. Few studies have considered the role that environmental exposure plays in the ...epidemiology of these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192 samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp. Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177; all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further, the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds in the 19th century.
Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The ...zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein‐coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST‐828 and ST‐1150 clonal complexes. The ST‐1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST‐828 complex bacteria have 10–11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross‐species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.
There has been an explosion of data describing newly recognized structural variants in the human genome. In the flurry of reporting, there has been no standard approach to collecting the data, ...assessing its quality or describing identified features. This risks becoming a rampant problem, in particular with respect to surveys of copy number variation and their application to disease studies. Here, we consider the challenges in characterizing and documenting genomic structural variants. From this, we derive recommendations for standards to be adopted, with the aim of ensuring the accurate presentation of this form of genetic variation to facilitate ongoing research.
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