Computer measurements and graphics of three-dimensional cellular ultrastructure

J O U R N A L OF L I L T R A S T R U C T U R E RESEARCH

75, 131-141 (1981)

Computer Measurements and Graphics of Three-Dimensional Cellular Ultrastructure PETER B . MOENS AND T. MOENS

Department of Biology, York University, Downsview, Ontario M3J 1P3, Canada Received August 12, 1980, and in revised form February 4, 1981 A relatively inexpensive system and simple procedures are described for storing on computer files structural information from serial electron micrographs. The use of such files is demonstrated for length and distance measurements in three dimensions, for computer drawings of the structures, rotation of the objects, for the construction of stereo images, and for the generation of cross-sectional views from longitudinal sections.

The availability of relatively inexpensive microcomputers and simple programming languages makes it feasible to utilize computer-assisted analysis of ultrastructure as a routine procedure (Stevens, 1977; Moens, 1978; Stevens, 1979). With a digitizer, a microcomputer, and a terminal, it is possible to measure distances, surfaces, and volumes in three dimensions. Computer-constructed images of cells and cell organelles can be produced with a cathode ray tube or a plotter. Excellent visualization of complex structures can be achieved through computer-drawn stereo pairs as has been shown for molecules such as transfer RNA (Sussman and Kim, 1976) and snake-venom neurotoxin (Tsernoglou et al., 1977). Display of structures can also be improved by "rotating" the original data to a favorable angle of view (McIntosh et al., 1979). It is not economical to use commercial computerized measurement units in this type of data storage, manipulation, and display since the unit would function only as a digitizer which by itself is a fraction of the cost of the whole unit. In addition the assembly described here has the capabilities of commercial units but is less expensive. It is the purpose of this paper to describe the equipment, the methods of data collecting, storage, and display, and to illustrate the use of this technology in some asp e c t s of cell biology. The n e c e s s a r y

computer programs are available from the authors. EQUIPMENT The diagram in Fig. 1 summarizes the equipment used for computerized measurements, data storage, manipulation, and display. The total cost need not exceed $10 000. Digitizer. We use a Numonics Model 1224 digitizer which includes the digitizing unit, a microcomputor with a display unit, and a keyboard (Numonics Corporation, 418 Pierce St., Lansdale, Pa. 19446, $4000). It has a resolution of 0.25 mm and a convenient freeranging cursor (60 x 60 cm) which can be used on any surface. The display console can be programmed with the keyboard to either display X - Y coordinates, pointto-point lengths, continuous lengths, or areas. The device will output the data to a computer by the "enter" command of the keyboard. The X - Y coordinates a r e sent over as a string of characters in a preferred form such as "_+2.251~_+9.171~1~ . . . " The use of the enter command prevents waste of memory space which happens when coordinates are recorded automatically at a high rate such as 100 coordinates per second. Instead, a structure such as shown in Fig. 2a can be circumscribed by 18 points (Fig. 2b) or 11 points (Fig. 2c) and a perfectly fitting computer drawing can be generated using the 18 points combined with a "smoothing" routine (Fig. 2d) (Rogers and Adams, 1976). Microcomputer. We use an Ohio Scientific Challenger III microcomputer (Ohio Scientific, 1333 South Chillicath Rd., Aurora, Ohio 44202) with two 8-in. floppy disks and a 48K random access memory ($4000). It is connected by T cable to the digitizer and the terminal (L.A. 36 DEC writer II, Digital Equipment Corp., Maynard, Mass. 01754, $1500) so that data can be entered simultaneously from both, an important feature in the tracing of a line in three dimen131 0022-5320/81/050131 - 11$02.00/0 Copyright (~ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

132

MOENS AND MOENS To record a linear structure passing through several sections, the X - Y coordinates for each point on the line are entered from the digitizer and the section number is typed in at the terminal giving a file of type

FIG. 1. Equipment used for computerized measurements and displays.

sions. Also, the terminal displays the data entered from the digitizer so that errors can be detected and corrected while digitizing. We use the BASIC programming language for its ease of operation, although other languages are available (about $500 each). With a BASIC compiler subroutines can be used but they are not necessary for the procedures reported here. Through a telephone coupler the system can be made to interact with larger computers and plotters. Plotter. Plotters are available from $1000 (Houston instruments HIPLOT, DMP-2). We use a Calcomp 81 (Calcomp, 2411 West LaPalma Ave., Anaheim, Calif. 92801, $5000), which is more complex than needed for the graphics shown here. It features eight pens, numerous operating modes, and an optional automatic drive.

METHODS

Electron Microscopy The tissues or cells are embedded in Epon or Spurr resin and sectioned on an ultramicrotome equipped with a diamond knife. Series of 20 to 40 sections are picked up from the water with a loop (we use a singlehole grid, 1 × 2 ram) and transferred to a Formvarcovered single-hole grid. After staining the material is examined with an electron microscope and a suitable cell, nucleus, or organelle is selected. Such a cell is then photographed consecutively in every section in which it appears (35-mm film) and printed on 8 × 10in. paper. The successive photographs are placed in proper alignment and a pair of corresponding fiducial marks is placed on each photograph of a series.

Tracing Depending on the type of structure to be recorded, different methods of tracing were used. Essentially linear structures such as microtubules or chromosome cores were traced throughout the series, one structure at a time. A final tracing was made from the preliminary tracing by drawing a line through the recorded points and marking where the line passes through the subsequent section (Fig. 3a). Structures with volume such as cells, mitochondria, nuclei, or chromosomes were recorded by tracing their outline in successive sections (Fig. 2).

X1Y1Z1 X2Y2Z2 XaY3Z3 X,Y,Z, where some of the Z values may be the same when a portion of the line curves in a given section. A scaling factor can be keyed in to digitize in inches, centimeters, or relative distances based on the magnification of the electron micrographs. To record the outline of a body in a given section, the fiducial marks of each tracing are matched with a set of marks on a fixed transparent overlay and a number of points on the outline are "entered" from the digitizer and the section number is typed in only once from the terminal. As a result, a file is produced for each section of the type X1Y~Z X2Y~Z X3Y3Z X,Y,Z. The process is repeated for each subsequent section.

Recording A sample BASIC program to record and store a line in three dimensions, such as the line shown in Fig. 3a, is as follows: 10 INPUT "TYPE IN A NAME FOR YOUR F I L E " ;F$ 20 OPEN " 0 " , I , F $ 30 I N P U T " T Y P E THE SECTION N U M BER"; Z 40 INPUT " E N T E R X - Y COORDINATES FROM DIGITIZER" ;A$ 50 X=VAL(MID$(A$, 1,6)) 60 Y=VAL(MID$(A$,7,10)) 70 PRINT #1,X,Y,Z 80 INPUT " L I N E FINISHED.'? V OR N";B$ 90 IF B $ = " Y " THEN 100 ELSE 30 100 END SAVE "RECORD3D" RUN The program is saved on disk under file name "RECORD3D". After the RUN command the program prompts the operator to give a file name, e.g., MT8 and to type the section number at the terminal and to enter the coordinates of a given point on the line from the digitizer. In line 70 the X - Y and Z values are stored on disk file MT8 and the next set of coordinates can be recorded after typing Y in response to line 80.

133

COMPUTER GRAPHICS OF ULTRASTRUCTURE

b

C

e

FIG. 2. Tracing and plots of contours. (a) O n e section of a series through a m e t a p h a s e c h r o m o s o m e . The length is about 5 t~m. (b) A c o m p u t e r - d r a w n plot using 18 points on the circumference of the c h r o m o s o m e . (c) S a m e as (b) but using only i I points. (d) S a m e as (b) but using a " s m o o t h " routine. (e) S a m e as (c) using a " s m o o t h " routine. T h e fit of (e) is not as good as that of (d). A plot of all contour lines is s h o w n in Fig. 6. The actual p r o g r a m we u s e is faster by omitting the instructions in lines 30, 40, and 80 once recording has started. T h e s a m e p r o g r a m can be modified to record the outlines of a structure in s u c c e s s i v e sections (Figs. 2, 6). Three-Dimensional Distances L e n g t h s of linear structures in the X - Y plane can be calculated and displayed directly by m o s t c o m m e r cial digitizers. T h e length of a line p a s s i n g t h r o u g h a n u m b e r of sections, h o w e v e r , requires the X, Y, and

Z coordinates of each point and it b e c o m e s n e c e s s a r y to interact the digitizer with a computer. T h e length can be c o m p u t e d while the coordinates are being digitized but it is m o r e a d v a n t a g e o u s to p u t the original data on file first and then u s e a separate p r o g r a m to carry out the calculations using the data on file. This way the calculations can be repeated for different section t h i c k n e s s e s or magnification scales. Also, the data can s u b s e q u e n t l y be u s e d for graphics display or other p u r p o s e s . A s a m p l e B A S I C p r o g r a m to calculate lengths in three d i m e n s i o n s from data on file is as follows:

134

MOENS AND MOENS

10

I N P U T " T Y P E IN THE N A M E OF THE DATA FILE";F$ 20 INPUT "TYPE IN THE SECTION THICKNESS IN uM";ST 30 INPUT " E N T E R THE DIGITIZED LENGTH OF A 1 uM L I N E " S C .% 1o

iS

"

". ~? •,~ *°°.

16 t6 a

b

40 OPEN " I " , I , F $ 50 INPUT #1,X1,Y1,Z1 60 IF EOF(1) THEN PRINT "THE LENGTH OF ";F$;" IS " ; S U M ; " uM" :END 70 INPUT #1,X2,Y2,Z2 80 X=(X1-X2)/SC: Y=(Y1-Y2)/SC: Z= (Z1-Z2)*ST 90 DIS=SQR(X*X+Y*Y+Z*Z) 100 S U M = S U M + D I S 110 XI=X2: Y I = Y 2 : Z I = Z 2 120 GOTO 60 Using the data file entered in the section "Recording," the operator would enter MT8 in response to line 10. If the section thickness is 80 nm, a value of 0.08 is entered at line 20. If the digitizer is reading in inches and a 1-/~m line is 2 in. long, then SC=2. In line 40, the disk file MT8 is opened and data can be INPUT " I " from the file to the memory. As the result of line 50, for example, X1 = 5.24, Y1 = 8.34, and Z1 = 15. Statement 60 checks if the END OF FILE (EOF) has been reached. If not, the next set of coordinates is read into X2, Y2, and Z2 at line 70. In line 80, the increment from one point to the next is calculated for X, Y, and Z and the values are in micrometers. The distance from one point to the next in three dimensions is computed in line 90 by DISTANCE = (X~+Y~+Z2)½. In line 100, the consecutive distances are summed as the program goes from point to point. In line 110, the second set of coordinates becomes the first set of coordinates and the program returns to line 60 and the next set of coordinates is read into X2, Y2, and Z2 and so on. When there are no more points left in the file, then line 60 will cause the printout of, for example, "THE LENGTH OF MT8 is 3.15 urn" and the program ends.

Plotting When the coordinates of a series of points, as in Fig. la, are on disk file, they can be used to produce a graphic display of the line they represent (Fig. 3b). The program required to produce the line depends on the type of equipment used. To emphasize the relative simplicity of the procedures, the program used in conjunction with the Ohio Scientific microcomputer and the Calcomp 81 plotter is listed below• Fro. 3. Tracings and computer plots of linear structures. (a) A pair of axial cores was traced through consecutive electron micrographs. The numbers correspond to the sections where the cores were observed. (b) A computer plot of the original data. (c) Same as (b) but using a "smooth" routine. (d) The same structure after 5° rotation around the Y axis; Figs. (c) and (d) are a stereo pair that, when viewed simuItaneously, demonstrates the three-dimensional configuration of the two axial cores (see also Fig. 5). The real distance between the two lines is about 0.1 /~m.

10 INPUT "TYPE THE FILE NAME TO BE PLOTTED" ;F$ 20 OPEN " I " , I , F $ 30 IF EOF(1) THEN END 40 INPUT #1,X,Y,Z 50 X$= STR$(INT(X* 100)) 60 Y$=STR$(INT(Y* 100)) 70 B $ = X $ + " / " + Y $ + " K I " 80 LPRINT B$ 90 GOTO 30

C O M P U T E R GRAPHICS OF U L T R A S T R U C T U R E

135

FIG. 4. Positions and distances in three dimensions. (a) Part of an Allium fistulosum meiotic prophase nucleus containing nucleoli (NO) and one centrornere (CE). The scale bar = 1/zrn. (b) and (c) A stereo pair of the nucleus reconstructed from 150 consecutive sections showing the positions of the eight paired centrorneres and the nucleoli. The contour lines of the nucleus were traced every 10th section and the centrorneres in every section. The three-dirnensional distances between the centrorneres are given in Table I.

136

MOENS AND MOENS

II

COMPUTER GRAPHICS OF ULTRASTRUCTURE In response to line 10 "type the file name to be plotted," the operator types in the name of the disk file that has the coordinates, for example, MT8. The file is opened for the input ( " I " ) into the memory in line 20 and one set of coordinates is put in memory at line 40. The next two lines change the X - Y values to the plotter scale and put them in character strings. Line 70 puts together a string of characters consisting of the X coordinate, a slash, the Y coordinate, the " K " command to move the pen to the point, and the " I " command for pen down on the paper. In the next line, the characters of that string are sent over to the plotter one by one. When all characters have been sent over, the program returns to line 30 and the next set of coordinates will be processed (if there are any left). Although X, Y, and Z are read in, only the X and Y coordinates are used for this two-dimensional plot. The lines in Fig. 3b were-produced with this program. The angular path of the line does not well represent the smooth curvature of the actual lines in the electron micrographs (Fig. 5a). The drawing can be improved by digitizing many more points or, more economically, by having the computer construct a curved path from one set of coordinates to the next. We use "An algorithm for generating cubic splines," a BASIC language smoothing routine from Rogers and Adams (1976) to produce Figs. 3c and d.

Rotation Once the coordinates of a linear structure of a body are on file, the structure can be rotated in space by rotating around the X, Y, or Z axis or a combination and then plotted in the new position. When rotating around the Y axis, only the X values change in the new plot. The change is a simple trigonometric function of the angle of rotation and the new X coordinate is given by XR = X*cos(B)/cos(A) where B and A are angles in radians relative to the Y axis of the old and new position ofX. A new file is created which contains the old Y values and the rotated X values. These are then plotted with the plotting program, The result of 45 ° rotation can be seen by comparing Fig. 6a with Fig. 6c.

137

Stereo Images By rotating the structure a moderate amount of about 5° to 7° and viewing the original and new images simultaneously, the three-dimensional configuration of the structure can be perceived. The fusion of the two images is facilitated by the use of a stereo viewer but can be attained unaided by staring at the images as if they are at a distance and then gradually focusing without altering the parallel alignment of the eyes. Large images can be viewed in stereo by crossing the eyes so that the left image is perceived by the right eye and vice versa. The stereo effect in this case is reversed. Examples are shown in Figs. 3c and d, 4b and c, 5b and c, and 6.

Artificial Cross Sections Once a structure or set of structures has been recorded and filed on disk, it becomes feasible to examine the structure in sectional views other than the original section. For example, if a spheroidal object (football shape) is sectioned longitudinally, eliptical outlines appear in the sections. A program can be written that will calculate the intersections of the recorded outlines with planes perpendicular to the longitudinal axis of the structure. The intersects are filed and later plotted as such or drawn with connecting lines to produce a cross-section view of the structure. In this example, a series of circles would be drawn which first increase in diameter and then decrease as the series of artificial cross sections goes from the center to the end of the spheroid. An example of spindle cross sections generated from a file of longitudinal sections is shown in Fig. 7. APPLICATIONS

D i s t a n c e s a n d P o s i t i o n s in Three D i m e n sions T h e d i s t r i b u t i o n o f d i s c r e t e s t r u c t u r e s in the cell such as granules, small vesicles, or nuclear pores,

can be quantified once the

X-Y

and Z coordinates

have

been

recorded

of each structure

from

serial electron

m i c r o g r a p h s . F o r e x a m p l e , i n a s t u d y o f interphase chromosome

movements

we used

F~o. 5. Positions and lengths in three dimensions. (a) One section through a yeast nucleus in meiotic prophase which contains 17 synaptonemal complexes (SC). The two dense lateral elements of each ribbonshaped SC were traced and digitized. (b) and (c) A stereo pair of all 17 SC. The attachments to the nuclear envelope are indicated by short lines across the ends of the SCs. Two of the SCs end in the nucleolus indicated by the dotted areas. The 3D lengths of all SCs are given in Table II. × 43 000.

138

MOENS AND MOENS

FIG. 6. Rotated display. (a) and (b) A stereo pair of all contours of internal structures of the metaphase I chromosome shown in Fig. 2. The internal structure (thick black lines) has the shape of a twisted eight so that the lower loop is in the plane of the paper and the upper loop is perpendicular to it. (c) and (d) A stereo pair after 45 ° rotation. The eight shape of the internal structure is more obvious from this viewpoint. FIG. 7. Artificial cross sections. (a) A longitudinal section of a mitotic spindle in the fungus Saprolegniaferax, with microtubules (MT) and spindle poles (SP). (b) Computer-drawn plot of all of the nonkinetochore microtubules of the above spindle. MTs originating at the pole on the left are in thick black lines, MTs from the right in medium black lines, and other MTs in fine black lines. Each MT was given a number and the numbers are printed at the ends of the MTs. The arrows indicate the positions of two artificial cross sections shown below. (c) and (d) Computer-generated cross sections of the spindle of positions indicated by arrows in (b). Squares represent MTs from the left, triangles MTs from the right, and others are marked by asterisks. × 36 000.

139

C O M P U T E R GRAPHICS OF U L T R A S T R U C T U R E

2

tl

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iii

d

la~ 5

22

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2

6~

FIGURE 7

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140

MOENS AND MOENS TABLE I

made from the X - Y and Z coordinates of the structure in consecutive sections. This technique has become particularly popular in the measurement of synaptonemal complexes as a means of chromosome identification and characterization at meiotic prophase (Gillies, 1972, 1973, 1979; Moens, 1973; Byers and Goetsch, 1975; Holm, 1977; Zickler, 1977; Carpenter, 1979). An example of plotting such linear structures is demonstrated in Fig. 5 and the length measurements are given in Table II. Each SC consists of two parallel cores which can be seen to twist and curve through the nuclear volume in the stereopair Figs. 5b and c. All ends are attached to the nuclear envelope, indicated by a short line across the end of each core. The two parallel cores of a given SC should, theoretically, be of equal length but slight variations in section thickness, shrinkage, and alignment cause differences in the lengths of core 1 vs core 2 of the SCs (Table II). In this case this is the only possible method of karyotyping this organism, the yeast Saccharomyces cerevisiae, because the chromosones are not visible in light microscopy.

DISTANCES IN THREE DIMENSIONS (IN /zm) BETWEEN THE EIGHT PARRS OF CENTROMERES IN AN Alliumfistulosum MEIOTIC PROPHASE NUCLEUS~ 1 --

2

3

4

5

6

7

8

7.1

3.3

7.5

7.4

4.4

10.7

7.6

1

--

5.2

4.4

1.6

8.1

5.3

6.3

2

--

4.3

5.3

5.5

8.4

5.4

3

--

4.0

8.9

6.3

5.2

4

--

7.7

4.0

4.9

5

--

9.6

6.0

6

--

5.1

7

--

8

a See Fig. 4. )~ = 6.05/zm, SD = 2.04.

the chromosomal centromeres (Fig. 4a) as recognizable markers of chromosome positions at different stages of the cell cycle (Church and Moens, 1976; Moens and Church, 1977). The positions of the eight centromeres in an Alliumfistulosum meiotic prophase nucleus are visualized in the computer-drawn stereopair of Figs. 4b and c. The degree of centromere congregation was determined from the distances between the centromeres (Table I) and their mean distance, J~, with standard deviation, SD. The results showed that in early interphase cells the mean was in the order of 2 to 3 Rotation /xm while in later stages it was about 6/xm, The purpose of rotating a structure is indicating a gradual dispersion of the cento view it from different vantage points tromeres. (Levinthal et al., 1974). Where the structure has a complex three-dimensional arLengths and Positions in Three Dimensions rangement, any one two-dimensional perLinear structures such as microtubules spective does not reveal all structural deor chromosomal cores seldom appear in tails. In the example below the structure of their entire length in a single section and interest has the configuration of a figure length measurement therefore can only be eight but the loops of the eight lie in planes T A B L E II THREE DIMENSIONAL LENGTHS (IN ~m) OF 17 SYNAPTONEMAL COMPLEXES IN A YEAST MEIOTIC PROPHASE NUCLEUS Synaptonemal complex n u m b e r 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Core 1 Core 2

2.5 2.6

2.3 2.4

2.3 2.2

2.0 2.0

2.0 1.9

2.0 1.9

1.8 1.7

1.6 1.6

1.5 1.5

1.4 1.5

1.4 1.2

1.2 1.2

1.1 1.3

1.1 1.2

0.7 0.7

0.6 0.7

0.6 0.6

Average

2.5

2.4

2.3

2.0

2.0

1.9

1.8

1.6

1.5

1.5

1.3

1.2

1.2

1.1

0.7

0.7

0.6

COMPUTER GRAPHICS OF ULTRASTRUCTURE

at 90° to each other. In Fig. 6a the lower loop (thick black lines) lies in the plane of the paper but the second loop lies perpendicular to the paper and therefore is difficult to visualize. After 45 ° rotation around the Y axis the lower loop has closed up somewhat but the upper loop has now opened and the figure eight configuration is more directly apparent. The stereo pairs confirm the three-dimensional arrangement of the eight and display the outer contours of the meiotic metaphase chromosome. The internal structure represents fine fibrillar material between sister chromatids (Fig. 2a) and it has the configuration of a classical three-chiasma bivalent (Moens and Church, 1979). Artificial Cross Sections It may be disirable to examine a structure in sectional planes other than the actual plane of sectioning. This is for example the case in the structural and functional analysis of the mitotic spindle. In longitudinal sections the linear continuity of the microtubules facilitates tracing, recording, and reconstructing the spindle but the information of interest, the interaction between the MTs (McIntosh et al., 1979; McDonald et al., 1979), is best viewed in cross-sectional displays of the spindle. These demands can be reconciled by computer-generated cross sections from data entered from a longitudinally sectioned spindle (Fig. 7). In the drawing of the original data (Fig. 7b), all MTs have been given a number and the ones from the left pole are drawn in thick black, the ones from the right in medium black, and the free MTs in thin black. The MTs can also be differentiated by the use of different colored pens or by the use of dotted and dashed lines. The structure of the spindle at two crosssectional planes is shown in Figs. 7c and d. The cross section in the left part of the spindle contains mostly MTs from the left pole

141

(squares) and only two MTs (22, 4, triangles) from the right pole are present. In the cross section to the right (Fig. 7d) there are fewer squares, more triangles, and two free MTs (11, 13, asterisks). The program can be extended to show where given MTs come within critical distance of other MTs. We wish to thank Dr. I. B. Heath for the Saprolegnia spindle material, C. E. Matthews for assistance with the assembly of the system, and the NSERC for financial assistance. REFERENCES BYERS, B., AND GOETSCH, L. (1975) Proc. Nat. Acad, Sci. USA 72, 5056-5060. CAm'ENTER, A. T. C. (1979) Genetics 92, 511-541. CrtURCH, K., AND MOENS, P. B. (1976) Chromosoma (Berlin) 56, 249-263. GILLIES, C. B. (1972) Chromosorna (Berlin) 36, 119130. GILLrES, C- B. (1973) Chromosoma (Berlin) 43, 145176. GILHES, C. B. (1979) Genetics 91, 1-17. HOLM, P. B. (1977) CarlsbergRes. Commun. 42, 103151. LEVlNTHAL, C., MACAGNO, E., AND TOUNTAS, C. (1974) Fed. Proc. 33, 2336-2340. McDoNALD, K. L., EDWARDS, M. K., AND MCINTOSH, J. R. (1979) J. Cell Biol. 83, 443-461. MCINTOSH, J. R., MCDONALD, K. L., EDWARDS, M. K., AND ROSS, B. M. (1979) J. Cell Biol. 83, 428442. MOENS, P. B. (1973) Cold Spring Harbor Syrup. Quant. Biol. 38, 99-107. MOENS, P. B. (1978) Int. Congr. Electron Microsc. 9th 3, 557-563. MOENS, P. l~., AND CHURCH, K. (1977) Chromosoma (Berlin) 61, 41-48. MOENS, P. B., AND CHURCH, K. (1979) Chromosorna (Berlin) 73, 247-254. ROGERS, D. F., AND ADAMS, J. A. (1976) Mathematical Elements for Computer Graphics, McGrawHill, New York. STEVENS, B, (1977) Biol. Cell. 28, 37-56. STEVENS, B. (1979) in FLEISCHER, S., AND PACKER, L., (Eds.), Methods in Enzymology, Vol. 56, pp. 718-728, Academic Press, New York. SUSSMAN, J. L., AND KIM, S.-H. (1976) Science •92, 853-858. TSERNOGLOU, D., PETSKO, G. A., MCQUEEN, J. E., JR., AND HERMANS, J. (1977) Science 197~ 13781380. ZICKLER, D. (1977) Chromosoma (Berlin) 61, 289-316.