Octane

BACs selected for cytogenetic mapping were colony-purified and end-sequenced to
verify clone identities (data not shown). BAC DNA was prepared by the standard
alkaline lysis method. To limit bias in the interpretation of FISH results, BAC
preparations were assigned a code number, and identical coded samples were delivered
to the Dimitri laboratory for mitotic FISH and the Zhimulev laboratory for polytene
FISH. The BAC FISH results were recorded without knowledge of the clone identities or
expected map locations.
FISH to mitotic chromosomes from the isogenized
y¹;
cn¹bw¹sp¹ reference strain (Brizuela et al. 1994 (link)) was performed as described
in Accardo and Dimitri (2010) (link).
FISH to polytene chromosomes was performed as described in Saunders (2004) (link) with modifications described here.
w^m4, SuUR
Su(var)3-9⁰⁶ larvae were grown at
25°C in uncrowded vials on standard fly food. Salivary glands were dissected in
Ephrussi-Beadle saline (Ephrussi and Beadle
1936) and fixed in a 3:1 mixture of ethanol and acetic acid for 30 min at
−20°C, squashed in 45% acetic acid, snap-frozen in liquid nitrogen, and
stored in 70% ethanol at −20^°C. Squashes of polytene
chromosomes were incubated in 2× SSC for 1 h at 65°C, washed three times
for 5 min in 2× SSC at room temperature, denatured in 2× SSC, 0.07 N NaOH
for 0.5 min, dehydrated in increasing concentrations of cold ethanol (70%, 80%, 100%)
for 3–5 min each, and air dried. DNA probes were labeled with biotin-16-dUTP
or digoxigenin-11-dUTP (Roche) in random-primed reactions with the Klenow fragment of
DNA polymerase I. Labeled probes were added to hybridization solution (50% formamide,
2× SSC, 10% dextran sulphate, 1.0% sonicated salmon sperm DNA) to a final amount
of 0.1–0.2 µg per slide. Hybridization was performed overnight at
37°C in a humid chamber. Unbound probes were removed with three 15-min washes in
0.2× SSC at 42°C. Slides were stained with avidin-FITC and rhodamine
anti-DIG conjugate in blocking solution (0.1% BSA, 1× DIG-blocking reagent
[Roche]) for 30 min at 37°C in a humid chamber and washed three times for 5 min
with 4× SSC, 0.1% Tween-20. Finally, 10 µl of antifade solution (2.5 mg/mL
of 1,4-diazobicyclo-[2.2.2]-octane in 2× SSC [Sigma]) with DAPI were added
before examination by fluorescence microscopy. In addition to BAC DNAs, marker gene
DNA probes (Supplemental Table S5) were used to correlate polytene regions with
mitotic regions.

In a 300-mL two-necked round-bottomed
flask equipped
with a magnetic stirring bar, a rubber septum and an argon balloon,
NaH (60% dispersion in mineral oil, 6.44 g, 161 mmol), and THF (60
mL) were added, respectively. The reaction mixture was stirred, and
octane-1,8-diol (3.92 g, 26.8 mmol) in THF (30 mL) was added. The
reaction mixture was warmed to reflux and stirred for 12 h. Then,
the reaction mixture was cooled to 0 °C and 2-bromohexanoic acid 5a (7.91 mL, 56.3 mmol) was added slowly to this reaction
mixture. The reaction mixture was warmed to reflux and stirred for
48 h. After the reaction, the reaction mixture was cooled to 0 °C
and ice water (100 mL) was added to quench the reaction. The whole
mixture was extracted with 1 M NaOH (5 × 10 mL). Then, the pH
was adjusted to 1 with 2 M H₂SO₄ solution. The
whole mixture was extracted with diethyl ether (5 × 10 mL). The
combined organic phases were washed with H₂O (20 mL), dried
(with Na₂SO₄), and concentrated in vacuo to
give a crude product. The crude product was purified by flash column
chromatography on silica gel (n-hexane/ethyl acetate/acetic
acid = 7:3:0.1) to give the title compound 7c (5.78 g).
In a 100-mL one-necked round-bottomed flask equipped with a magnetic
stirring bar and a rubber septum, product 7c, EtOH (15
mL), and MeSNa aq (15 wt %, 15 mL) were added, respectively. The reaction
mixture was warmed to 40 °C and stirred for 24 h. After the reaction,
the reaction mixture was concentrated in vacuo, and the pH was adjusted
to 1 with 2 M H₂SO₄ solution. The whole mixture
was extracted with diethyl ether (5 × 10 mL). The combined organic
phases were washed with H₂O (20 mL), dried (with Na₂SO₄), and concentrated in vacuo to give the title
compound 7c (5.41 g, 54%). Yellow oil; ¹H
NMR (500 MHz, CDCl₃) δ 3.86 (dd, J = 4.8, 7.1 Hz, 2H), 3.59 (dt, J = 6.5, 9.4 Hz,
2H), 3.49–3.37 (m, 2H), 1.82–1.72 (m, 4H), 1.65–1.56
(m, 4H), 1.46–1.29 (m, 16H), 0.91 (t, J =
7.0 Hz, 6H); ¹³C{¹H} NMR (126 MHz, CDCl₃) δ 177.5, 78.8, 71.0, 32.2, 29.5, 29.1, 27.1, 25.8,
22.3, 13.8; IR (neat) 3424, 2952, 2866, 1643, 1459, 1383, 1279, 1207,
1128, 926, cm^–1; HRMS (EI) m/z: [M-CHO₂]⁺ calcd for C₁₉H₃₇O₄ 329.2692, found 329.2692.