Table 1.

Characteristics of transgenic lines expressing wild-type (PwMhc2) and mutant (PwMhcR759E) myosin

		Flight testing (at 2 days/7 days)
Line name (chromosome location)	Myosin level ± SE	Number tested	% Up	% Horizontal	% Down	% Not at all	Flight index ± SE
PwMhc2 (X)	1.00±0.03	116/197	57.8/45.7	14.6/26.4	15.5/22.8	12.1/5.1	4.4±0.1a/4.2±0.1
PwMhcR759E-V8 (X)	0.98±0.04	98/114	0/0	27.5/0	40.9/1.8	31.6/98.2	1.3±0.2/0.03±0.01
PwMhcR759E-7B (3)	0.97±0.04	91/111	0/0	21.9/0	40.7/2.7	37.4/97.3	1.6±0.3/0.05±0.01
PwMhcR759E-V5 (X)	0.95±0.03	111/ND	0/ND	19/ND	46.8/ND	34.2/ND	1.6±0.3/ND

^aFlight index data for 2-day-old PwMhc2 are from Kronert et al.³⁵

The R759E mutant Mhc gene was constructed using the wild-type genomic construct PwMhc2.³⁶ The 12.5-kb Eag I restriction fragment was subcloned into pBluescriptKS (Stratagene, La Jolla, CA), which had been digested with Eag I. The resulting subclone, p3’Mhc was digested with Xba I and Spe I. The 160-bp fragment from this digest (containing the R759 coding region) was subcloned into pBluescriptKS that had been digested with Xba I and Spe I. The resulting subclone, pR759, was subjected to site-directed mutagenesis using the QuickChange II kit (Stratagene) and the exon specific-primer 5′-CCCGATATGTACGAAATTGGTCACACC-3′ containing the R759E nucleotide coding change (bold). Upon sequence confirmation of the R759E site-directed mutagenesis product, the pR759E subclone was digested with Xba I and Spe I. The resulting 160 bp fragment was used to replace the wild-type Xba I - Spe I fragment of p3’Mhc. The resulting clone was digested with Eag I, and the Eag I fragment was replaced back into the wild-type construct PwMhc2 at its Eag I site. Ligation sites were confirmed by DNA sequencing, as were all splice junctions and coding regions of the final pwMhcR759E plasmid. BestGene, Inc. (Chino Hills, CA) produced transgenic lines by P element-mediated transformation³⁷ and mapped chromosome locations by standard genetic crosses using balancer chromosomes. Transgenes were crossed into the Mhc¹⁰ (null for myosin in the indirect flight muscle) background.¹⁴ To confirm the expression pattern of PwMhcR759E and to insure that no wild-type copy of Mhc was present in PwMhcR759E transgenic line, we used exon-specific primers and RT-PCR as previously described.³⁵ RNA was isolated from upper thoraces of two-day-old female adult flies of wild type (yw) and PwMhcR759E. Restriction enzyme digests and sequence analysis of the RT-PCR products from PwMhc2R759E compared to yw confirmed there was no difference in alternative exon usage and that the mutagenized codon encoding R759E was present in PwMhcR759E but not in yw (data not shown). Myosin accumulation relative to actin was determined by SDS polyacrylamide gel electrophoresis. Dissected upper thoraces were homogenized in 50 μl SDS gel buffer from 5 two-day-old female flies. Eight μl of sample were loaded on a 9% polyacrylamide gel. Each transgenic line was analyzed five times, each time with a freshly prepared sample. Coomassie blue stained gels were digitally scanned and protein accumulation was determined using NIH image software. Mean values are compared to the wild-type transgenic control, PwMhc2. Flight assays were performed with two-day-old and one-week-old adult female flies at 22°C. Flight was assessed by the ability to fly up (U), horizontal (H), down (D) or not at all (N) when released in a plexiglass box with a light source at its top.³⁸ The flight index is defined as: 6U/T + 4H/T + 2D/T + 0N/T; T is the total number of flies tested.³⁹