Analysis of Sequence Data 1 Computer-Aided Analysis

The sequence data was initially loaded into an Apple Macintosh and analysed using the DNA Strider and Gene Jockey programs. These were used to detail overlaps from different sequencing reactions, generate translations in all reading frames, calculate amino acid compositions and molecular weights of the derived proteins, print-out hydropathy plots (Kyte & Doolittle 1982), simple alignments and dot-plot matrices. Hydropathy plots were calculated using the values shown in the table (Table 3).

Table 3. Hydropathic Index of Amino Acids.

Amino Acid Index Value

Ala (A) 1.8 Arg(R) -4.5 Asp (D) -3.5 Asn (N) -3.5 Cvs (C) 2.5 Glu (E) -3.5 Gln(Q) -3.5 Gly(G) -0.4 His (H) -3.2 he a) 4.5 Leu (L) 3.8 Lys (K) -3.9 Met (M) 1.9 Phe (F) 2.8 Pro(P) -1.6 Ser(S) -0.8 ThrCT) -0.7 Trp (W) -0.9 Tyr(Y) -1.3 Val(V) 4.2

Additional sequence analysis and database searches were performed using the Intelligenetics Corporation suite of programs (licenced to ICRF London) via a VAX workstation. SwissProt, NIH GenBank, EMBL and NBRF FIR databases were searched with the programs FASTDB, IFIND and BIFIND (Wilbur & Lipman 1983; Lipman & Pearson 1985; Gribskov et al 1987).

2.23.2 p i Calculation

It is possible, knowing the amino acid composition, to estimate the pi of a protein by statistically averaging the pKr values of all the charged amino acids. An estimate of the value can be made from the equation:

pl = ^ ( ( ^ X pKj^i) + (jc X pK^2) + (x X pK^g) + ...+ (x X pK^j))

where pK^ is the dissociation constant of the side chain group for amino acids 1 to i, jc is

the number of such amino acids in the total protein and n is the total number of amino acid types being considered.

Table 4. Amino Acid Dissociation Constants (Values from Lehninger 1970).

Amino Acid _PKr D 3.86 E 4.25 H 6 . 0 C 8.33 Y 10.07 K 10.53 R 12.48 C terminal average 2.185 N terminal average 9.549 2.24 Genome Analysis 2.24.1 DNA Preparation

High molecular weight genomic DNA was isolated from freshly dissected tissues using a procedure modified from Blin & Stafford (1976). 0.5-0.7cm^ pieces of tissue were placed in 1.5ml eppendorf tubes containing 700pl extraction buffer (50mM Tris-HCl pH8, lOOmM EDTA, lOOmM NaCl, 1% SDS) finely minced using small dissecting

scissors, 35|il lOmg/ml proteinase K (Life Technologies) added, mixed by repeated inversion and incubated at 55°C for 16 hours. Undigested tissue was pelleted by centrifugation at 13,000g for 5 minutes and the supernatant transferred, using a wide bore pipette tip to a clean 1.5ml tube containing 25pi lOmg/ml RNAase (Worthington Enzymes), mixed and incubated at 37°C for 2 hours. The solutions were first extracted with equal volumes of phenol, then twice with a phenol / chloroform mixture and once with chloroform alone.

0.7 volumes of isopropanol (BDH) were carefully layered onto the preparations and high molecular weight DNA spooled onto sealed capillary tubes at the interphase. Each capillary tube carrying DNA was sequentially washed in 70% and 100% ethanol (BDH), gently air dried and placed overnight in 500[xl TE (7.6). The concentration and integrity of DNA was analysed both by agarose gel electrophoresis and OD readings.

2.24.2 Genomic DNA Digestion

Restriction endonuclease digestions of l-10|ig (as required) were carried-out in lOOpl volumes, with a 2-5 fold excess of restriction enzyme at the required incubation temperature usually for 16 hours (Section 2.13.2). Successful digestion was confirmed by agarose gel electrophoresis (Section 2.14) of aliquots of each digest withdrawn before the addition of 0.1 volumes of 3M sodium acetate pH5.2, 2 volumes of absolute ethanol and incubation at -85°C for 16 hours. Precipitated DNA was centrifuged at 13,000g for 20 minutes at 4°C, supernatants were discarded and the pellets resuspended in IxTBE.

2.24.3 Electrophoresis

Dissolved DNA was mixed with loading buffer, incubated at 56°C for 5 minutes, chilled on ice, loaded onto a 0.9% HOT agarose gel and run at 15V (constant voltage) for 16-20 hours. Genomic digests and XHindlll MW markers (IBI) were visualised in Ipg/ml ethidium bromide on a UV transilluminator and photographed with a transparent ruler as a scale.

2.24.4 Blotting

Pretreatment of gels for vacuum blotting was conducted outside of the Hybaid vacuum blotting apparatus. Blotting onto nylon filters (Hybond N+ Amersham) was performed as described (Section 2.15(b)). The filters were either alkali fixed (0.4M NaOH) for 20 minutes or air dried and baked at 80°C for 2 hours.

2.24.5 Genomic Southern Blot Hybridisation

Filters were soaked in 6xSSC until thoroughly wetted then immersed for 2 minutes

before transfer to 50ml prehybridisation buffer (Table 1) at 60°C for two hours. a32p labelled probe at 5x10^ - IxlO^cpm/pg was heated to 100°C for 5 minutes, chilled on ice for 5 minutes and added to the filters in 20ml fresh prehybridisation buffer at 60°C for 24 hours. Probe was discarded and the filters washed three times for 5 minutes each in 200ml

of 2xSSC, 0.1% SDS at 60°C, autoradiographed, rewashed to IxSSC, 0.1% SDS at 60°C and autoradiographed (Section 2.20(a)).

2.24.6 C ross-species H ybridisations

Filters were floated on 6xSSC until thoroughly wetted then immersed for 1 minute.

Prehybridisation was performed in 7% SDS, 1% BSA and 0.5M sodium phosphate buffer pH7.2 at 50°C for 24 hours. Hybridisation was performed in the same buffer at 50°C for 48-72 hours. Filters were washed twice in 200ml 2xSSC at 50°C for 30 minutes each, autoradiographed then rewashed in the same buffer at 60°C and autoradiographed. Where required, further washes in 2xSSC, 0.1% SDS at 70°C then IxSSC, 0.1% SDS at 70°C were performed.

Chapter 3 Results

Immunofluorescence and immunoblot data demonstrate that the upper isoform of the C4 doublet (transgelin) is an actin-associated polypeptide that is transformation sensitive (Shapland et al 1988). Since examination of the literature suggested that transgelin (C4^) was one of only a limited number of actin-binding proteins that were transformation sensitive (Shapland et al 1993) an obvious need therefore existed for the elucidation and analysis of its primary structure.