Molecular weight estimates of the fiber precursors show that there are two copies of P34 in the A half-fiber and two copies of P37 in each of the other half-fibers. The major experimental advantage of T4 is that the availability of mutants in the morphogenetic genes allows experimental disruption of the assembly process. Instead, the sequence represents control of the order of interaction of the gene products themselves.
The isolated A-half fiber carries a knob at one end, which is also visible at one end of the whole fiber. This step does not affect the antigenic properties of the structure, but is necessary to allow interaction with the A-half fiber. The total purification was 25- to 100-fold, depending on the specific activity of the crude lysate.
The homogeneity of fraction III preparations was checked by electrophoresis on polyacrylamide gels at pH 4.3 and 9.5 (panel I). The results for fraction III BC' are shown in panel I e. for C and BC show that the major band on each of the pH 4.3 gels is the antigen fiber structure. Two observations indicate that these free half-fibers are not products of the breakdown of the whole fiber, but rather.
A SBP
Second, by repeated DEAE chromatography of the ABC preparation, all contaminating BC' could be removed. at 4 to 6 °C and rechromatographed, free BC' no longer appeared. These observations indicate that the coupling of BC' and A to form ABC is not readily reversible under our conditions. d) Electron microscopy characterization of (i) C, BC and BC'. No differences were observed between fraction III and fraction IV BC fibers (not shown).
Histograms of the length distributions of the three structures are shown in Figures 3 a, b and c, respectively. As an additional check on this result, the length distribution of an equal mixture of C and BC' was determined (Fig. 3 d). It is seen to be distinctly bimodal, with the means corresponding to the lengths of C and BC'.
LENGTH A
GENETIC CONTROL OF THE MAJOR TAIL FIBER POLYPEPTIDES
Purified preparations of whole T4 bacteriophage, tail fiberless particles, whole tail fibers, and four tail fiber precursors were dissociated by brief heating at 100°C in 1% sodium dodecyl sulfate. Analysis of the dissociated structures by polyacrylamide gel electrophoresis in the presence of SDS and mercnptoethanol revealed two high molecular weight polypeptides (150,000 and 123,000 daltons) as major tail fiber components. The larger of the two was missing from extracts from cells infected with gene 34 amber mutants, and the smaller from extracts from cells infected with gene 37 amber mutants.
Molecular weight calculations indicate that two copies of each polypeptide are present in each complete tail fiber. By scintillation counting of the radioactive material in the P34 and P37 bands after gel fractionation, gene 36 amber mutations were shown to reduce the synthesis of P37, implying that genes 36 and 37 are included in a single transcription unit. Bacteriophage T4 provides an excellent system to study genetic control of the assembly of a complex biological structure.
In order to understand the molecular mechanisms of gene control of some assembly steps, we decided to study tail fiber assembly in detail. The first paper in this series (King & Wood, 1969) described the sequence of interaction of gene products in the composition of tail fibers and identified several fiber precursors. This study continues the characterization of isolated fibers by determining their subunit structure by dissociation and polyacrylamide gel electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate (SDS).
The genes controlling the synthesis of the two major polypeptides are identified and the assembly of these polypeptides into half-fibers is examined by comparing their solubility and resistance to dissociation before and after assembly. Methods for the preparation and purification of fiberless phage and tail particles are given in Dickson, Barnes & Eiserling, (1970). b) Purified precursors of tail fibers and whole fibers. Four tail fiber precursors and whole fibers were prepared from lysates of mutant-infected cells as described by Ward et al.
Defective lysates will be indicated by the number of the defective gene, e.g. d) Electrophoresis on polyacrylamide gels containing SDS. Purified phage and purified fiberless particles were dissociated in SDS, electrophoresed on SDS gels, and then stained and destained.
ABC +
New bands representing the amber peptides will appear, but may be obscured by the other bands in the lower region of the gels. Both bands 19.0 and 21.0 are present in these lysates, indicating that these genes do not greatly affect the synthesis of the P34 and P37. The identity of the peaks of counts corresponding to P34 and P37 was established by comparison with the count of a parallel gel of a mixture qf 14.
The results are shown in Table 3 as the percentage of P34 and P37 counts found in the supernatant fractions of the different lysates. The role of genes 57 and 38 in the assembly of the tail fibers is not available for direct investigation, because 57- and 38-defective extracts. The determination of the half fiber molecular weights indicated that P34 and P37 are present in two.
If so, P34 must change conformation during dimerization in order to explain the lack of antigenicity of the P34 monomer. Unfortunately, temperature-sensitive mutations are not available in genes 57 or 38, so the reversibility of P34 and P37 aggregation is not easily tested. Antigen B, which results from the action of gene 36 on the C fiber, is located at the end of the C fiber which interacts with half of the A fiber.
In the lower branch of the pathway, P37 with a molecular weight of 123,000 dimerizes to form the C antigen and the C half fiber under the control of genes 57 and 38. Arrows indicate the assembly steps under the control of the numbered genes shown above them. The results in Part II show that P34 and P37 are the major polypeptides of the complete fiber.
In part II it was argued that the formation of the A and C antigens could involve conformational changes in P34 and P37. To test this, comparative studies of the conformations of the polypeptides in structures c, BC and BC' could be performed. The methods developed in this work should help determine the role of the genes involved in gene assembly.
Care must be taken to collect the extruded gel at the bottom of the vials, as it stubbornly adheres to surfaces.
WIRE
SUPPORTS
PLUNGER
After electrophoresis, the gel, in its tube, is placed in a V-shaped trough and a clamped plunger is inserted into the top of the gel. Then the fractions are obtained by turning the fixing, which is connected to the piston by means of a screw (1 turn . ; 1.4 nun). The slice is attached to the knife blade, which is immersed in a scintillation vial containing counting fluid.
Large quantities of the counting cocktail can be prepared by mixing anunonium hydroxide with NCS until the solution is clear). and then adding the mixture to toluene scintillation. The entire cocktail can be used for at least a week, stored at room temperature or better 4-6°, in the dark. After collection of fractions, the vials are stoppered and allowed to stand at room temperature for 2 hours or overnight. see below) with occasional shaking.
Tritium counting efficiency, as determined by adding a known number of 3H-toluene (NEN) to the counting cocktail, was 38"/o. Gel slices obtained with a miniature gel extruder and slicer showed little variation: slices from uniformly labeled of the gel with 3H gave a 2% standard deviation. Since extruding gels of standard size through the needle could cause distortion of the labeling patterns, the resolution of the separated protein bands after fractionation was measured.
FRACTION NUMBER 2b
STAI NED GEL
The gel was fixed, stained with Coomassic Brilliant Blue and destained according to Ward's method. The protein peaks are not distorted by the fractionator and there is little tail counting. The fractionation shows that the peak of the 3H counts migrates one fraction before the peak of 14c.
In both cases, decanting the scintillation cocktail from the gel and recounting showed that the labeled protein had diffused out of the gel and into the scintillation cocktail. For fractionation of uniformly labeled gels of standard size, the sum of the counts from all fractions is 96 ± 3% of the counts in the gel before polymerization for both 14c and 3H. This may be due to the dye quenching the counts or loss of protein during staining and destaining.
The use of NH 4 OH in the counting cocktail reduces light-induced phosphorescence of the NCS-toluene scintillation mixture. 3 Without NH 40H, samples with less than a few hundred counts of 3H must be stored overnight in the dark to allow the phosphorescence to decay. With Nti40H in the cocktail, a small amount of phosphorescence decays to background within a few minutes.
The resolution of the two closely spaced bands after fractionation is not as high as when the protein bands are stained in the unfractionated gel. Almost all procedures require gel digestion to achieve good count yields. We believe this is due to efficient diffusion of protein and water from the gels, possibly aided by partial hydrolysis of peptide bonds in the gels.
The diffusion of proteins out of the gel is certainly accelerated by having the gel macerated with extrusion fractionator, but the recovery of counts is also quantitative with standard size or miniature gels fractionated by excision if the fractions are allowed to stand overnight before counting. The method provides high counting efficiency, does not require hydrolysis of the gel before counting and allows collection of gel fractions directly in scintillation vials.
