Transcription Unit Of Gene Research Articles

The complete plastid genome and nuclear ribosomal transcription unit sequences of <i>Spiraea prunifolia</i> f. <i>simpliciflora</i> (Rosaceae)

<i>Spiraea prunifolia</i> f. <i>simpliciflora</i> Nakai is a perennial shrub widely used for horticultural and medicinal purposes. We simultaneously obtained the complete plastid genome (plastome) and nuclear ribosomal gene transcription units, 45S nuclear ribosomal DNA (nrDNA) and 5S nrDNA of <i>S. prunifolia</i> f. <i>simpliciflora</i>, using Illumina short-read data. The plastome is 155,984 bp in length with a canonical quadripartite structure consisting of 84,417 bp of a large single-copy region, 18,887 bp of a short single-copy region, and 26,340 bp of two inverted repeat regions. Overall, a total of 113 genes (79 protein-coding genes, 30 tRNAs, and four rRNAs) were annotated in the plastome. The 45S nrDNA transcription unit is 5,848 bp in length: 1,809 bp, 161 bp, and 3,397 bp for 18S, 5.8S, and 26S, respectively, and 261 bp and 220 bp for internal transcribed spacer (ITS) 1 and ITS 2 regions, respectively. The 5S nrDNA unit is 512 bp, including 121 bp of 5S rRNA and 391 bp of intergenic spacer regions. Phylogenetic analyses showed that the genus <i>Spiraea</i> was monophyletic and sister to the clade of <i>Sibiraea angustata</i>, <i>Petrophytum caespitosum</i> and <i>Kelseya uniflora</i>. Within the genus <i>Spiraea</i>, the sections <i>Calospira</i> and <i>Spiraea</i> were monophyletic, but the sect. <i>Glomerati</i> was nested within the sect. <i>Chamaedryon</i>. In the sect. <i>Glomerati</i>, <i>S. prunifolia</i> f. <i>simpliciflora</i> formed a subclade with <i>S. media</i>, and the subclade was sister to <i>S. thunbergii</i> and <i>S. mongolica</i>. The close relationship between <i>S. prunifolia</i> f. <i>simpliciflora</i> and <i>S. media</i> was also supported by the nrDNA phylogeny, indicating that the plastome and nrDNA sequences assembled in this study belong to the genus <i>Spiraea</i>. The newly reported complete plastome and nrDNA transcription unit sequences of <i>S. prunifolia</i> f. <i>simpliciflora</i> provide useful information for further phylogenetic and evolutionary studies of the genus <i>Spiraea</i>, as well as the family Rosaceae.

Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts.

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.

Homology-based reconstruction of regulatory networks for bacterial and archaeal genomes

Gene regulation is a key process for all microorganisms, as it allows them to adapt to different environmental stimuli. However, despite the relevance of gene expression control, for only a handful of organisms is there related information about genome regulation. In this work, we inferred the gene regulatory networks (GRNs) of bacterial and archaeal genomes by comparisons with six organisms with well-known regulatory interactions. The references we used are: Escherichia coli K-12 MG1655, Bacillus subtilis 168, Mycobacterium tuberculosis, Pseudomonas aeruginosa PAO1, Salmonella enterica subsp. enterica serovar typhimurium LT2, and Staphylococcus aureus N315. To this end, the inferences were achieved in two steps. First, the six model organisms were contrasted in an all-vs-all comparison of known interactions based on Transcription Factor (TF)-Target Gene (TG) orthology relationships and Transcription Unit (TU) assignments. In the second step, we used a guilt-by-association approach to infer the GRNs for 12,230 bacterial and 649 archaeal genomes based on TF-TG orthology relationships of the six bacterial models determined in the first step. Finally, we discuss examples to show the most relevant results obtained from these inferences. A web server with all the predicted GRNs is available at https://regulatorynetworks.unam.mx/ or http://132.247.46.6/.

Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters.

To successfully engineer mammalian cells for a desired purpose, multiple recombinant genes are required to be coexpressed at a specific and optimal ratio. In this study, we hypothesized that synthetic promoters varying in transcriptional activity could be used to create single multigene expression vectors coexpressing recombinant genes at a predictable relative stoichiometry. A library of 27 multigene constructs was created comprising three discrete fluorescent reporter gene transcriptional units in fixed series, each under the control of either a relatively low, medium, or high transcriptional strength synthetic promoter in every possible combination. Expression of each reporter gene was determined by absolute quantitation qRT-PCR in CHO cells. The synthetic promoters did generally function as designed within a multigene vector context; however, significant divergences from predicted promoter-mediated transcriptional activity were observed. First, expression of all three genes within a multigene vector was repressed at varying levels relative to coexpression of identical reporter genes on separate single gene vectors at equivalent gene copies. Second, gene positional effects were evident across all constructs where expression of the reporter genes in positions 2 and 3 was generally reduced relative to position 1. Finally, after accounting for general repression, synthetic promoter transcriptional activity within a local multigene vector format deviated from that expected. Taken together, our data reveal that mammalian synthetic promoters can be employed in vectors to mediate expression of multiple genes at predictable relative stoichiometries. However, empirical validation of functional performance is a necessary prerequisite, as vector and promoter design features can significantly impact performance.

Chromatin-based mechanisms to coordinate convergent overlapping transcription.

In eukaryotic genomes, the transcription units of genes often overlap with other protein-coding and/or noncoding transcription units1,2. In such intertwined genomes, the coordinated transcription of nearby or overlapping genes would be important to ensure the integrity of genome function3-6; however, the mechanisms underlying this coordination are largely unknown. Here, we show in Arabidopsis thaliana that genes with convergent orientation of transcription are major sources of antisense transcripts and that these genes transcribed on both strands are regulated by a putative Lysine-Specific Demethylase 1 family histone demethylase, FLOWERING LOCUS D (FLD)7,8. Our genome-wide chromatin profiling revealed that FLD, as well as its associating factor LUMINIDEPENDENS9, downregulates histone H3K4me1 in regions with convergent overlapping transcription. FLD localizes to actively transcribed genes, where it colocalizes with elongating RNA polymerase II phosphorylated at the Ser2 or Ser5 sites. Genome-wide transcription analyses suggest that FLD-mediated H3K4me1 removal negatively regulates the transcription of genes with high levels of antisense transcription. Furthermore, the effect of FLD on transcription dynamics is antagonized by DNA topoisomerase I. Our study reveals chromatin-based mechanisms to cope with overlapping transcription, which may occur by modulating DNA topology. This global mechanism to cope with overlapping transcription could be co-opted for specific epigenetic processes, such as cellular memory of responses to the environment10.

The repertoire of ABC proteins in Clostridioides difficile

ATP-binding cassette (ABC) transporters belong to one of the largest membrane protein superfamilies, which function in translocating substrates across biological membranes using energy from ATP hydrolysis. Currently, the classification of ABC transporters in Clostridioides difficile is not complete. Therefore, the sequence-function relationship of all ABC proteins encoded within the C. difficile genome was analyzed. Identification of protein domains associated with the ABC system in the C. difficile 630 reference genome revealed 226 domains: 97 nucleotide-binding domains (NBDs), 98 transmembrane domains (TMDs), 30 substrate-binding domains (SBDs), and one domain with features of an adaptor protein. Gene organization and transcriptional unit analyses indicated the presence of 78 ABC systems comprising 28 importers and 50 exporters. Based on NBD sequence similarity, ABC transporters were classified into 12 sub-families according to their substrates. Interestingly, all ABC exporters, accounting for 64% of the total ABC systems, are involved in antibiotic resistance. Based on analysis of ABC proteins from 49 C. difficile strains, the majority of core NBDs are predicted to be involved in multidrug resistance systems, consistent with the ability of this organism to survive exposure to an array of antibiotics. Our findings herein provide another step toward a better understanding of the function and evolutionary relationships of ABC proteins in this pathogen.

Design, validation and evaluation of a SYBR green-based quantitative PCR array for comprehensive analysis of adenovirus type 5 transcriptional patterns

The adenoviral genome encodes coordinately expressed early and late gene transcriptional units that specify a complex collection of extensively spliced overlapping mRNAs. These complexities confound the generation of compatible, validated and optimized qPCR assays that permit comprehensive evaluation of adenoviral transcription. We have developed and evaluated a compilation of qPCR assays that represent the majority of the human adenovirus 5 (hAdV5) genome and allow for absolute and relative quantification of transcriptional activity. A panel of specific adenovirus gene primer pairs was designed through computational modeling to be compatible under a single reaction condition, precisely amplify spliced transcript products within each gene class, and not result in cellular or viral RNA/DNA background amplification. Primer pairs and reaction conditions were optimized to generate a single amplification product that was specific for its target amplicon with minimal intra-assay variability. The specificity of target amplicons was confirmed by dissociation curve analysis, gel electrophoresis and sequencing. In all, thirty-two primer sets representing specific gene products, as well as, pan early and late gene regions were validated under identical amplification conditions, thereby enabling a comprehensive assessment of adenoviral transcription within a single plate array. In order to generate positive control templates and to facilitate absolute quantification of gene expression, all target amplicons were cloned to create gene target-specific standards. These plasmid amplicon controls demonstrated that the SYBR qPCR assays exhibited optimal amplification efficiencies with a high sensitivity of detection to less than 10 copies and a linear amplification across at least eight orders of magnitude. The effectiveness and utility of the comprehensive adenoviral transcriptional array was assessed by investigating the changes in Ad5Wt gene expression at 72 versus 24 h post infection. Predictably, overall gene expression was globally increased at 72 h post infection; however, levels of E2 and Late transcripts exhibited the greatest increased expression, reflecting their necessity at this time point for genomic replication and virion assembly. Taken together, these data demonstrate that the adenoviral qPCR transcriptional array is a modular, scalable, and cost-effective method to comprehensively and accurately assess hAdV5 gene transcription. This array is broadly applicable to facilitate: adenoviral vector development; assessment of cell complementation of knockout viruses; antiviral mechanism of action evaluation; next-generation sequencing data validation.

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ54 factor RpoN2 regulated bacterial virulence on rice in a flagellum‐independent manner in Xoo. In this study, both yeast two‐hybrid and pull‐down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two‐component system PilS‐PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT‐PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2‐binding sequence GGN10GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT‐PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2‐PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo.

STARR‐seq and UMI‐STARR‐seq: Assessing Enhancer Activities for Genome‐Wide‐, High‐, and Low‐Complexity Candidate Libraries

The identification of transcriptional enhancers and the quantitative assessment of enhancer activities is essential to understanding how regulatory information for gene expression is encoded in animal and human genomes. Further, it is key to understanding how sequence variants affect enhancer function. STARR‐seq enables the direct and quantitative assessment of enhancer activity for millions of candidate sequences of arbitrary length and origin in parallel, allowing the screening of entire genomes and the establishment of genome‐wide enhancer activity maps.In STARR‐seq, the candidate sequences are cloned downstream of the core promoter into a reporter gene's transcription unit (i.e., the 3′ UTR). Candidates that function as active enhancers lead to the transcription of reporter mRNAs that harbor the candidates’ sequences. This direct coupling of enhancer sequence and enhancer activity in cis enables the straightforward and efficient cloning of complex candidate libraries and the assessment of enhancer activities of millions of candidates in parallel by quantifying the reporter mRNAs by deep sequencing. This article describes how to create focused and genome‐wide human STARR‐seq libraries and how to perform STARR‐seq screens in mammalian cells, and also describes a novel STARR‐seq variant (UMI‐STARR‐seq) that allows the accurate counting of reporter mRNAs for STARR‐seq libraries of low complexity. © 2019 The Authors. Basic Protocol 1: STARR‐seq plasmid library cloning Basic Protocol 2: Mammalian STARR‐seq screening protocol Alternate Protocol: UMI‐STARR‐seq screening protocol—unique molecular identifier integration Support Protocol: Transfection of human cells using the MaxCyte STX scalable transfection system

A Split Staphylococcus aureus Cas9 as a Compact Genome-Editing Tool in Plants.

Split-protein methods—where a protein is split into two inactive fragments that must re-assemble to form an active protein—can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (_430N/431C and _739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9_739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9_739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

RNA Polymerase II Subunits Exhibit a Broad Distribution of Macromolecular Assembly States in the Interchromatin Space of Cell Nuclei

Nearly all cellular processes are enacted by multi-subunit protein complexes, yet the assembly mechanism of most complexes is not well understood. The anthropomorphism "protein recruitment" that is used to describe the concerted binding of proteins to accomplish a specific function conceals significant uncertainty about the underlying physical phenomena and chemical interactions governing the formation of macromolecular complexes. We address this deficiency by investigating the diffusion dynamics of two RNA polymerase II subunits, Rpb3 and Rpb9, in regions of live Drosophila cell nuclei that are devoid of chromatin binding sites. Using FRAP microscopy, we demonstrate that both unengaged subunits are incorporated into a broad distribution of complexes, with sizes ranging from free (unincorporated) proteins to those that have been predicted for fully assembled gene transcription units. In live cells, Rpb3 exhibits regions of stability at both size extremes connected by a continuous distribution of complexes. Corresponding measurements on cellular extracts reveal a distribution that retains peaks at the extremes but not in between, suggesting that partially assembled complexes are less stable. We propose that the broad distribution of macromolecular species allows for mechanistic flexibility in the assembly of transcription complexes.

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS.

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP C -deficiency

The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP(C)). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP(C)-like function to maintain the viability of Prnp(0/0) mice lacking the PrP(C) protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a "C1" C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, "N1," which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg1 gene transcription unit that overlaps the Sprn 3' UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn(0/0) mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp(0/0) embryos using lentiviruses targeted against the Sprn 3' UTR, we established that double-knockout mice deficient in both Sho and PrP(C) are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP(C) and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP(C), we infer that Sho's activity will prove germane to the maintenance of neuronal viability in postnatal life.

Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons

The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.

Genetic variability of transcript abundance in pig peri-mortem skeletal muscle: eQTL localized genes involved in stress response, cell death, muscle disorders and metabolism

BackgroundThe genetics of transcript-level variation is an exciting field that has recently given rise to many studies. Genetical genomics studies have mainly focused on cell lines, blood cells or adipose tissues, from human clinical samples or mice inbred lines. Few eQTL studies have focused on animal tissues sampled from outbred populations to reflect natural genetic variation of gene expression levels in animals. In this work, we analyzed gene expression in a whole tissue, pig skeletal muscle sampled from individuals from a half sib F2 family shortly after slaughtering.ResultsQTL detection on transcriptome measurements was performed on a family structured population. The analysis identified 335 eQTLs affecting the expression of 272 transcripts. The ontologic annotation of these eQTLs revealed an over-representation of genes encoding proteins involved in processes that are expected to be induced during muscle development and metabolism, cell morphology, assembly and organization and also in stress response and apoptosis. A gene functional network approach was used to evidence existing biological relationships between all the genes whose expression levels are influenced by eQTLs. eQTLs localization revealed a significant clustered organization of about half the genes located on segments of chromosome 1, 2, 10, 13, 16, and 18. Finally, the combined expression and genetic approaches pointed to putative cis-drivers of gene expression programs in skeletal muscle as COQ4 (SSC1), LOC100513192 (SSC18) where both the gene transcription unit and the eQTL affecting its expression level were shown to be localized in the same genomic region. This suggests cis-causing genetic polymorphims affecting gene expression levels, with (e.g. COQ4) or without (e.g. LOC100513192) potential pleiotropic effects that affect the expression of other genes (cluster of trans-eQTLs).ConclusionGenetic analysis of transcription levels revealed dependence among molecular phenotypes as being affected by variation at the same loci. We observed the genetic variation of molecular phenotypes in a specific situation of cellular stress thus contributing to a better description of muscle physiologic response. In turn, this suggests that large amounts of genetic variation, mediated through transcriptional networks, can drive transient cell response phenotypes and contribute to organismal adaptative potential.

The genomes and comparative genomics of Lactobacillus delbrueckii phages

Lactobacillus delbrueckii phages are a great source of genetic diversity. Here, the genome sequences of Lb. delbrueckii phages LL-Ku, c5 and JCL1032 were analyzed in detail, and the genetic diversity of Lb. delbrueckii phages belonging to different taxonomic groups was explored. The lytic isometric group b phages LL-Ku (31,080 bp) and c5 (31,841 bp) showed a minimum nucleotide sequence identity of 90% over about three-fourths of their genomes. The genomic locations of their lysis modules were unique, and the genomes featured several putative overlapping transcription units of genes. LL-Ku and c5 virions displayed peptidoglycan hydrolytic activity associated with a ~36-kDa protein similar in size to the endolysin. Unexpectedly, the 49,433-bp genome of the prolate phage JCL1032 (temperate, group c) revealed a conserved gene order within its structural genes. Lb. delbrueckii phages representing groups a (a phage LL-H), b and c possessed only limited protein sequence homology. Genomic comparison of LL-Ku and c5 suggested that diversification of Lb. delbrueckii phages is mainly due to insertions, deletions and recombination. For the first time, the complete genome sequences of group b and c Lb. delbrueckii phages are reported.

SATB2 haploinsufficiency in patients with cleft palate

Abstract De novo translocation interrupting the transcription unit of SATB2 gene has been associated with cleft palate only (CPO). We tested for the presence of the copy number of SATB2 gene in a sample of 92 patients with CPO using a quantitative real-time PCR approach. In one patient (1%, 95% CI = 0.2%–6%), a 19 Mb de novo deletion encompassing the SATB2 gene was detected. These results suggest that SATB2 gene deletions do not play an important role in the etiology of cleft palate.

Chromatin structure is implicated in "late" elongation checkpoints on the U2 snRNA and beta-actin genes.

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the beta-actin gene, where a "late" P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and beta-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.

Fibroblast-Derived Induced Pluripotent Stem Cells Show No Common Retroviral Vector Insertions

Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.