After starting the program, DownArrow and RightArrow Keys are effective:

RightArrow toggles cursor speed with display of sums of RNA or enzyme.

DownArrow gets bottom line menu.

F1-F8 gets access to variable settings for Operon I - Operon VIII

F9 gets access to variables common to all operons and global settings of same variable on all operons with one exception; proteolysis of gene product from OP 8 (if active) can be accessed from right column.

F10 closes program

F11 - (reserved)

F12 displays peak intervals (replaced by distance to saddle point if existent), cell cycling time (if cell cycle is running), and subsequently (by striking any letter key) peak heights of RNA and enzyme-pathway in the selected number of operons, provided more than one cell cycle with at least one altered variable has been run. RNA deriving from first operon is only shown in a contact-inhibited cell. From F12 screen, source curves are still accessible by UpArrow, then immediately DownArrow to freeze image whereupon F12 may again be pressed. Pressing only UpArrow causes program to resume and recompute at the most recent cell cycle

Shift+Escape clears altered settings and reopens 1:st cell cycle (Ver. 1.0 only)

Shift+UpArrow while cell cycle is running or in bottom line menu clears screen and replots graphs while maintaining altered settings.

General: Fractions are a factor by which the compound is multiplied in each of the 30-200 computation loops comprising the cell cycle. Fraction values less than 1.0 should generally be used except in the case of steady-state level of GAP where values close to and around 1 may be used.

1. Effective Decay of Gene-Activating Principle: The factor 'A' in time-dependent exponential decay (exp (-At/M)) of inducer of prior operon that induces (or stimulates) transcription in menu operon

2. Steady State -level of Gene-Activating principle (GAP) -inducer: The factor 'M' in exponential decay (exp (-At/M)) of the compound that induces (or stimulates) transcription in menu operon. Numerical values more than 1.0 represent levels of GAP above steady state (equivalent of slower effective decay).

3. GAP-specific protease breaks down gene product of prior operon (sensitive, alter moderately!). Not that this variable pertaining to gene products of last operon, OP-n, is accessible in menu OP-(n+1) except when there are 8 active operons, in which case it is accessible in the All-Operon Menu (F9)!

5. GAP-threshold is minimum amount of gene product of prior operon required to cause transcription of menu operon. Only the amount of GAP above the threshold is seen by transcribing enzymes.

6. Restriction enzymes (RNASes) are active on RNA of menu operon only, before the RNA has been used for translation.

7. Post-ribosomal RNASes are active on RNA of menu operon only, after the RNA has been used for translation

8. Threshold of amount of RNA of menu operon above which it is detected by ribosomes. Only the amount of RNA above the threshold is seen by the ribosomes.

9. Gene amplification of inducing (=prior) operon, integers ≥ 1, only

10. Gene amplification of menu operon, integers ≥ 1, only

11. Competitive RNA-inhibition in menu operon represents specific competition on this RNA by other RNA (This is not the same as constitutive RNA, see below)

General: During contact inhibition, the operon which normally induces the trigger gene for commitment (= menu operon) also back-induces an even more prior operon than itself. In the case of commitment regulated on the 3:rd operon this means the gene product of the 2:nd operon induces transcription of the 1:st operon with maintained effectiveness even though its function as inducer of the trigger gene for commitment is suppressed. The rationale for emulating contact inhibition in this manner is that differentiated functions should be enhanced in contact-inhibited cells. The operons prior to the trigger operon for commitment thus also represent differentiated functions besides representing cell cycling pathways. The back-inducing capability may be accessed through the second column of input values in the OP3 and OP5 menus (starting with a parenthesis). The values in parenthesis are not effective unless the cell is contact-inhibited - quiescent. All parameters requiring active cell metabolism, such as RNASes and proteases, are shared with OP3 or OP5 and the back-inducing operon; only the parameters regulating transcription, such as effective decay of GAP, steady state level of GAP, and gene amplification, are separate. In the case that contact-inhibition is regulated on the 5:th operon, the gene product of the 4:th operon may by choice induce the 1:st, the 2:nd, or the 3:rd operon in this manner (default is 1:st). If the cross-induction is on the 2:nd or the 3:rd operon, that operon becomes amplified by a factor of 2 during contact-inhibition. Constant expression of differentiated functions may be obtained by cross-inducing the immediate prior operon of the inducing operon. Stretching the cross-induction further backwards causes oscillations of expression, which is a consequence of repetitively switching the back-induction on and off. In contact-inhibited cells, peak values refer to mean values of sometimes oscillating differentiated functions. The magnitude of oscillation may be diminished by increasing the half-life of mRNA in the OP1 (or OP2 or OP3) and the All-OP menus. The trigger mechanism for contact inhibition is when the level of expression of the gene (OP2 or OP4) prior to the trigger gene (OP3 or OP5) is above a threshold written in the OP3 or OP5 menus and the level of expression of the trigger gene itself is less than 3. The rise of the OP2 or OP4 expression following OP3 or OP5 suppression, which is inherent in this gene kinetics, may be a consequence of competition of mRNA for precursors, gene-regulatory factors, transport mechanisms, ribosomal sites and/or other factors. The level of expression of the inducing gene of OP2 and OP4 (i.e. OP1 or OP3) is also important. The threshold is adjustable in the OP3 or OP5 menus.

1b. Effective Decay of Gene-Activating Principle: (duplicate of above)

2b. Steady State -level of Gene-Activating principle (GAP) -inducer: (duplicate of above)

9b. Gene amplification of inducing (=prior) operon (duplicate of above)

12. Enzyme-pathway threshold of gene product of prior operon above which contact inhibition becomes effective may be altered by reference to "most recent peak value" of this gene product.

13. Differentiated functions starting at operon (I, II, or III). This is only available in case the contact inhibition is regulated at the 5:th operon.

14. Forced differentiation is a time lag in the number of computation loops before differentiated functions in a contact inhibited cell are again made silent. As of date of writing, this has not been made functional.

15. Effective decays of all gene-activating principles

16. Gene-activating principles below or above steady state

17. Unspecific proteases acting on all enzymes (sensitive)

18. Thresholds of GAPs before pathways can be transcribed

19. Unspecific nuclear RNASes (sensitive)

20. Unspecific post-ribosomal RNASes (sensitive)

21. Thresholds before RNAs are detected by ribosomes

22. Overall gene amplifications, integers ≥ 1

23. Constitutive (silent) RNA in denominator of RNA-pool sent to ribosomes

24. Anabolic capacity of ribosomes common to all RNAs

25. Mitosis emulation by maintaining a portion (fraction) of last expressed enzyme-pathway into the next cell cycle

26. Mitosis emulation by further keeping a fraction of RNA of last expressed gene into the next cell cycle (zero by default)

1. The program has been made for altering numerical values of the input variables after completion of each cell cycle. It may be advantageous to let the output values stabilize by running two cell cycles before inspecting the output or before again altering the input variables. A stabilized cell cycle is seen when the bottom line displays the text "CECY #n" and the graphs are steadily being plotted followed by display of the bottom line text "All results for this cell cycle have been compiled". While the latter text is shown one may access the bottom line menu by pressing the down arrow.

2. The program re-calculates the pitch of the x-axis if any cell cycle becomes longer than any of the previous ones. In such a case, it is recommended to wait until the display has been stabilized before reading the output or before altering the variables again.

3. The program has no error handling routines for "unlikely" values of the input variables. Use likely and reasonable values only! Excessively high peak heights are ignored, they are not visible, and they may cause the program to crash. Very low peak heights are ignored, especially in contact-inhibited cells. If the cell runs low on products from the last gene expressed and the cell is not contact-inhibited there might not be sufficient inducers for the next cell cycle and all gene expression will fade away until the program crashes because of math errors.

4. Should the program hang, it is always possible to press Alt+Line Feed and then close the program window by clicking on the X-button in the upper right corner. The program sometimes hangs because the MS-DOS memory for keyboard input has been corrupted. Usually, restarting the program is sufficient for restoring its functionality but in exceptional circumstances it might be necessary to restart the computer.

5. A minor nuisance is when the input line has fallen below the uppermost line when opening an operon menu. In such a case, use empty line feeds until the last line has been input (but not longer) and then reopen the same operon menu!

6. The operon menus ignore zeros in order to speed up operation by making plain line feed an escape sequence. In order to set a zero value for a parameter it is necessary to enter a comparatively low value close to zero, for example 0.001.

Except for the equation that describes the synthesis of mRNA, mitosis emulation, and emulation of contact inhibition-quiescence (cf. above), all computed metabolic processes are equivalent of trivial exponential decays. However, the equation describing the synthesis of mRNA first reaches a peak and then levels off exponentially as a function of amount of gene-activating principle (GAP). The reason for this behavior is that the GAP is broken down by its own pathway. The approach to a maximum value is important for obtaining a constant average level of expression in a contact-inhibited cell. In a cycling cell, competition between the various species of continuously degraded mRNA contribute to the decline of the early mRNA species. The manner of approaching the maximum value of mRNA synthesis may be evaluated by substituting the default values of the original equation, thereby changing the core theory. This feature is available from the first menu in the program.

Graphics from the screen may be stored by first pressing Alt+Line Feed keys, then the "PrintScreen" key and then pasting into an image processing program like MS Paint ® or MS Photo Editor ®. Some further image processing using "cut" and "paste" is necessary to get only the graph in the picture file. The same experiment may then be resumed after again pressing the Alt+Line Feed keys (Win 2000). Other operative systems may have particular features that require particular procedures. If these are applicable to Version 1.0 they should also be applicable to Version 5.0.

The program makes extensive use of memory available to MS-DOS (or MS-DOS emulator). Local settings that restrict the memory available to MS-DOS may hamper the functionality of the program. In such a case it is advisable to learn how to alter the memory settings of MS-DOS (MS-DOS emulator) on the particular local operative system and try to run the program with some kind of expanded memory. Version 5.0 requires an accessible C:/ catalogue.

15 consecutive cell cycles can be studied without re-opening the program. mRNA contents and gene expression deriving from the up to 7-8 operons are displayed continuously. Phase lengths and peak values for the up to 15 cell cycles can also be displayed.

At any time during a session, the variable settings for a sequence of cell cycles can be made permanent whereupon settings in additional cell cycles may be cancelled to restore the most recent memorized setting.

Shift+Z restores the most recent memorized setting.

Shift+X prior to operon menus erases only the most recent cell cycle (even while running; waiting for the completion of a cell cycle is not necessary).

On closing the program the session's variable settings (last saved and most recent) may be stored for future use. In such a case the vertical axis is re-scaled in the next session.

The start-up settings of the variables may be altered to start from most recent cell cycle or last saved cell cycle in prior session. In opening menus,

0+LF opens cell cycle with standard settings of all variables,

1+LF selects last cecy in most recent run if the data were actively saved in prior session,

2+LF selects last saved cecy in most recent run if the data were actively saved in prior session.

Version 5.0 prints out numerical results in ".txt" -files from where the data may be accessible to other graphics programs, for example using "macros". The macros are not provided. The ".txt"-files (RNA and pathway in separate files) represent a) screen display of cell cycle, b) screen display of peak intervals and peak heights, and c) cell cycle transcription and expression of individual operons in every cell cycle, each in a separate file. The graphics program to which the data is written must be capable of extrapolating over occasionally left-out coordinate values.

Version 5.0 is installed by dragging program icons to a target folder (Win 2000) and then running a short installation program that merely creates a couple of additional folders and files. Other operative systems may have other methods of transferring the files from the diskette or the CD to the target folder. The installation of Version 5.0 and 1.0 does not rely on registry editing or any other programmed manipulation of the operating system. Uninstalling is done by merely dragging or sending the files and folders to the computer's paper bin.

Several improvements of imperfections in Ver. 1.0 have been done in Ver. 5.0. Some improvements could not be done already in Version 1.0 because they would increase the risk of RAM shortage and were impossible or not easily done within the one-exe-file format of Version 1.0.

Information about access to and availability of Version 5.0 is provided in the info menu of Version 1.0.

The program was developed using DOS on a Windows platform. It also runs on a DOS-emulator under Linux. Ver. 1.0 has been tested for basic functionality with RedHat 8.0 and the binary distribution of Dosemu 1.2.2-1with freedos from dosemu.org. In this environment the graphics unfold in the DOS window and it is not necessary to press Alt + Shift to go to a whole screen. It runs considerably more rapidly and with some processors it may be necessary to slow down speed by editing the dosemu-freedos environment.

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