1H, 15N, and 13C chemical shift assignments of mouse HOXA13 DNA binding domain

The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of many important proteins during embryonic morphogenesis. We report complete NMR chemical shift assignments of the mouse HOXA13 DNA binding domain (A13DBD; BMRB no. 16252)

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

Backbone chemical shift assignments of mouse HOXA13 DNA binding domain bound to duplex DNA

The homeobox gene (Hoxa13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of many important proteins during embryonic morphogenesis. We report complete backbone NMR chemical shift assignments of mouse Hoxa13 DNA binding domain bound to an 11-residue DNA duplex (BMRB no. 16577)

Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to <i>N</i>‑Debenzoyl‑<i>N</i>‑(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase

In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in <i>Taxus</i> plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale <i>in vitro</i> biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product <i>N</i>-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to <i>N</i>-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis–Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue <i>N</i>-debenzoyl-<i>N</i>-(2-furoyl)­paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile <i>N</i>-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the <i>N</i>-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective <i>O</i>- and <i>N</i>-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis

<i>LncRNA-HIT</i> Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes

<div><p>Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), <i>LNCRNA-HIT</i> in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, <i>LncRNA-HIT</i> was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of <i>LncRNA-HIT</i>-p100/CBP complexes by ChIRP-seq revealed <i>LncRNA-HIT</i> associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by <i>LncRNA-HIT-</i>p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in <i>LncRNA-HIT</i> or p100 transcripts revealed a significant decrease in expression of many of the <i>LncRNA-HIT</i>-associated loci. <i>LncRNA-HIT</i> siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the <i>LncRNA-HIT</i> siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that <i>LncRNA-HIT</i> is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using <i>LncRNA-HIT</i> and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.</p></div

Expression analysis of the lncRNA <i>LncRNA-HIT</i>.

<p><b>(A)</b><i>In situ</i> hybridization using an antisense <i>LncRNA-HIT</i> riboprobe detects the transcript as early as E 10.5 in the distal limb which expands throughout the limb bud at E 11.5 <b>(B)</b>. <b>(C)</b> <i>In situ</i> hybridization using a sense orientation <i>LncRNA-HIT</i> riboprobe detects no <i>LncRNA-HIT</i> transcripts, confirming the unidirectional transcription of <i>LncRNA-HIT</i> in the same orientation as the 5’ HoxA genes in the developing limb. <b>(D and E)</b> Analysis of <i>Hoxa13</i> expression shows a high level of overlap with <i>LncRNA-HIT</i> in the distal limb. <b>(F-H)</b> Analysis of <i>Hoxa9-Hoxa11</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i> in the limb bud. <b>(I-K)</b> Analysis of <i>Hoxd11-Hoxd13</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i>. <b>(L)</b> <i>LncRNA-HIT</i> is also expressed in the developing genital tubercle, gut epithelium, urogenital sinus, spinal cord, and vertebral bodies at E 13.5. <b>(M-N)</b> <i>LncRNA-HIT</i> expression is detected in the digit perichondrial tissues, digit joint fields, and in the developing carpal/tarsal skeletal elements in E 13.5 forelimbs and hindlimbs. <b>(O)</b> Relative fold expression of <i>LncRNA-HIT</i> and <i>Hottip</i> expression in the E 11.5 limbs as determined by qRTPCR. Values represent the <i>Gapdh</i> normalized average expression of <i>LncRNA-HIT</i> and Hottip in the limb calculated from three independent analyses. Bars represent the standard deviation of the mean from the three independent assays. UGS = urogenital sinus, VB = vertebral body, GT = genital tubercle, GE = gut epithelium, SC = spinal cord.</p

siRNA-mediated reduction in <i>LncRNA-HIT</i> or <i>Snd1</i> results in reduced levels of 5’ HoxA gene expression.

<p><b>(A)</b> Relative gene expression after transfection with <i>LncRNA-HIT</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test. <b>(B)</b> Relative gene expression after transfection with <i>Snd1</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test.</p