Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the ...specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.
Display omitted
•We constructed a GFP fusion protein with Heliothis virescens pheromone gland Fatty Acyl Reductase.•The enzyme was localized in the endoplasmic reticulum.•The N-terminal of the enzyme is important for organelle targeting and function.•A Fluorescent Protease Protection-assay revealed a C-terminal towards the cytoplasm.•The endoplasmatic reticulum is proposed to be an important site for moth pheromone biosynthesis.
As part of the 2002 Shelf-Basin Interactions (SBI) process study, measurements of the seasonal variation in the export flux of particulate organic carbon (POC) are reported for the upper waters of ...the Chukchi Sea. POC fluxes were quantified by determination of
234Th/
238U disequilibrium and POC/
234Th ratios in large
(
>
53
μ
m
)
aggregates collected using in situ pumps. Samples were collected at 35 stations on two cruises, one in predominantly ice-coved conditions during the spring (May 6–June 15) and the other in predominantly open water during the summer (July 17–August 26). Enhanced particle export was observed in the shelf and slope waters, particularly within Barrow Canyon, and there was a marked increase in particle export at all stations during the summer (July–August) relative to the spring (May–June).
234Th-derived POC fluxes exhibit significant seasonal and spatial variability, averaging
2.9
±
5.3
mmol
C
m
-
2
d
-
1
(
range
=
0.031
–
22
mmol
C
m
-
2
d
-
1
)
in the spring and increasing
∼
4
-fold in the summer to an average value of
10.5
±
9.3
mmol
C
m
-
2
d
-
1
(
range
=
0.79
–
39
mmol
C
m
-
2
d
-
1
)
. The fraction of primary production exported from the upper waters increases from
∼
15
%
in the spring to
∼
32
%
in the summer. By comparison, DOC accumulation associated with net community production represented
∼
6
%
of primary production
(
∼
2
mmol
C
m
-
2
d
-
1
)
. The majority of shelf and slope stations indicate a close agreement between POC export and benthic C respiration in the spring, whereas there is an imbalance between POC export and benthic respiration in the summer. The implication is that up to
∼
20
%
of summer production
(
∼
6
±
7
mmol
C
m
-
2
d
-
1
)
may be seasonally exported off-shelf in this productive shelf/slope region of the Arctic Ocean.
The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the ...Frontabdominal-7 cis-regulatory region (Fab-7') dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-7' is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-7' deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste' allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor.
Infant acute lymphoblastic leukemia (ALL) with KMT2A‐gene rearrangements (KMT2A‐r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard ...next‐generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A‐r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A‐r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer‐associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most‐targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low‐frequency PI3K/RAS mutations without a higher‐frequency signaling mutation. Further, drug‐resistant clones with FLT3D835H or NRASG13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A‐r ALL harbor low‐frequency cancer‐associated mutations, implying a vast subclonal genetic landscape.
Fatty-acyl CoA reductases (FAR) convert fatty acids into fatty alcohols in pro- and eukaryotic organisms. In the Lepidoptera, members of the FAR gene family serve in the biosynthesis of sex ...pheromones involved in mate communication. We used a group of closely related species, the small ermine moths (Lepidoptera: Yponomeutidae) as a model to investigate the role of FARs in the biosynthesis of complex pheromone blends. Homology-based molecular cloning in three Yponomeuta species led to the identification of multiple putative FAR transcripts homologous to FAR genes from the Bombyx mori genome. The expression of one transcript was restricted to the female pheromone-gland tissue, suggesting a role in pheromone biosynthesis, and the encoded protein belonged to a recently identified Lepidoptera-specific pgFAR gene subfamily. The Yponomeuta evonymellus pgFAR mRNA was up-regulated in sexually mature females and exhibited a 24-h cyclic fluctuation pattem peaking in the pheromone production period. Heterologous expression confirmed that the Yponomeuta pgFAR orthologs in all three species investigated Y. evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner) encode a functional FAR with a broad substrate range that efficiently promoted accumulation of primary alcohols in recombinant yeast supplied with a series of biologically relevant C14- or C16-acyl precursors. Taken together, our data evidence that a single alcohol-producing pgFAR played a critical function in the production of the multicomponent pheromones of yponomeutids and support the hypothesis of moth pheromone-biosynthetic FARs belonging to a FAR gene subfamily unique to Lepidoptera.
The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines ...its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.
Fab-7 deletions in the bithorax complex have a novel gain-of-function phenotype, typically transforming parasegment 11 (PS11) into PS12 identity. Genetic analysis indicates that removal of the Fab-7 ...element results in the fusion of the iab-6 (PS11) and iab-7 (PS12) cis-regulatory domains into a single regulatory domain that inappropriately regulates Abdominal-B in PS11. This has led to the hypothesis that Fab-7 is a chromatin domain boundary that normally functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains. We use several different enhancer blocking assays to demonstrate that Fab-7 has the insulating properties expected of a domain boundary. We define a minimal fragment of Fab-7 sufficient for enhancer blocking, and demonstrate that it is completely distinct from an adjacent Polycomb-dependent silencer. We compare Fab-7 to the su(Hw) insulator element, and show that Fab-7 enhancer blocking activity is intermediate between that of five and twelve reiterated binding sites for the Su(Hw) protein. These results support the model that Fab-7 functions as a domain boundary within the context of the bithorax complex, making Fab-7 one of the first boundary elements that is known to have an essential function in vivo.
PDF
