If within the lozenge functional unit, the arrangement is:       A                              1_z^g                                B
                                                                                         a               .                    1_z^s                            b

(1_z^g to the left and 1_z^s to the right), the outside marker arrangement would be small a to the left and large B to the right.
 

        This is a very important point. Draw this out and you should see it. The wildtype sequence at the 1_z^g position must come from the bottom chromosome and the wildtype sequence at the 1_z^s position must come from the top chromosome. If the relative positions of 1_z^g and 1_z^s are switched (1_z^g to the right and 1_z^s to the left), then the opposite outside marker arrangement (A, b) would be found in the wildtypes.

         Subsequent work showed that the results from lozenge locus were not exceptional. Identical results were found with the waxy locus of maize, and the rosy locus of Drosophila, just to name some of the early classics.
 

        So, the take-home lesson from this is that while the genetic unit defined by function is almost the same as that defined by recombination, the fact that rare recombinational events can occur within the unit of function makes the two units different. That recombination occurs within the unit of function makes the unit of recombination smaller than the unit of function.
 

        Another point, so far not mentioned, but which now should be comprehensible, is that the unit of mutation ( the element that changed) must also be smaller than the unit of function. Were this not the case, recombination to "remove" these elements from the mutant gene to produce the wildtype gene would not be possible. This is clarified below.

Of most importance, the fact that recombination could occur within the gene (as defined by the test of function)  meant that the gene could by "split".   Mendel taught us that the gene was particulate and hence could be examined as a single entity whereas subsequent studies showed that  one could actually study parts of the gene.  The  parallel between "splitting" the gene and "splitting" the atom was not lost on physicists. 

        The work of physicist, turned geneticist, Seymour Benzer greatly clarified how the gene, as a unit of function, could be described in subunits of mutation and recombination. You should appreciate, at this junction, the fact that recombination within the unit of function - or gene as we now think about it - is a rare event. Hence, experimentally, what was needed was a mechanism to quickly identify rare recombinational events. Benzer fine tuned a system whereby only the rare events survived and he did it in an organism that was (1) quite small and (2) had a very short generation time. This is described below:

     But first some terms as defined by Benzer and some classic papers:
 
 

The unit of recombination is the smallest element in the one-dimensional array that is interchangeable (but not divisible) by genetic recombination-Recon.

The unit of mutation is the smallest element that when altered, can give rise to a mutant form of the organism -Muton.

        [Terms not used much today. These are equivalent to the nucleotide pair.]
 

The unit of function is defined by mutants, which when present in the trans arrangement, lead to a defective phenotype (mutant) but when present in the cis arrangement give rise to wild type. Such mutants are said to belong to the same cistron. The cistron then is a unit defined by the relationship between two mutants.
 

Cis and trans clearly have their roots in chemistry. "Cistron" then is derived from a combination of cis and trans.
 

What is a gene?
 

Benzer, S. 1957. "The Elementary Units of Heredity" in The Chemical Basis of Heredity. ed.       McElroy and B. Glass        (Baltimore, The John Hopkins Press ).    This paper is on file and is required reading.  If you understand the material presented in class, this should be no problem.

Benzer, S. 1955. Fine structure of a genetic region in bacteriophage. Proc. Natl. Acad Sci 41:344 -354
.
Benzer. S. 1959. On the topology of the genetic fine structure.  Proc. Natl. Acad Sci 45:1607 -20.

 Benzer, S. 1961. On the topography of the genetic fine structure. Proc. Natl. Acad Sci 47:403-415.

  Benzer, S. 1962. Sci. Amer. 206:70.

  Hayes, W. The Genetics of bacteria and their viruses. 2nd Edition.

   Pontecorvo, G. 1952. Adv. in Enzymology 13:121.

  What is an operon?

Jacob, F and J Monod.  1961.  Genetic regulatory mechanisms in the synthesis of proteins.  J. Mol. Biol. 3: 318-356.  This paper is on file and is required reading.  It gives a good overview of gene expression and enzyme regulation  at the time and places the concept of the gene and its expression in the context as described in class.)

Pardee, Jacob and Monod. 1959. J. Mol. Biol. 1:165 .

Jacob and Monod. 1961. Cold Spring Harbor Symposium. 26:

The lactose operon. Ed. by Beckwith, J.R. and D. Zipser. 1970. CSHL

Reznikoff. 1972. Ann. Review Genetics. 6: 133-152.
 

        Benzer greatly clarified these units with his work with the bacterial phage T4. Phage are bacterial viruses. They infect cells and can take over the cellular mechanism, replicate and make more phages. In doing so, they kill the cell and when the cell lyses or burst, progeny viruses are released. Neighboring cells are infected and the cycle is repeated. If one has a plate of bacteria growing and places one virus particle in the plate, one cell is infected and killed. Then neighboring cells are infected and the cycle is repeated. Within hours, this can be seen as a pinhole in the plate. The hole is called a plaque. The size of the plaque increases with time.
 

Benzer isolated mutants in which the size of this hole was altered. Timing was off. These mutants gave large plaques and they were termed rapid lysis (r).
 

                                  Bacteria

phage                       B
wild type                    wild

rII                               r

        Mutants seen on B E. coli were at a selective disadvantage. Thus it was impossible to measure, in quantitative terms, the number of wildtype and the number of mutants in a population by using this E. coli host. (However, as you will note below, quantitative data were actually not used much by Benzer.)   However on E. coli S (sometimes called KI2 in early papers), both genotypes grew at the same rate and, in fact, had indistinguishable phenotypes. In other words, the sizes of the plaques were the same.
 

  Phage                     S (K12)

wild type                         wildtype

rII                                   wildtype

        He also noted that on E. coli K lambda ( l ) mutants did not grow at all. This is very important since it provides a tremendous selection scheme. If a population plated out on K l is composed of all mutants, then no plaques are produced. However, if the population contains some rare wildtypes, they are easily seen and isolated since they produce plaques.
 

So, all together:
 

             Phage                                                               Bacteria
 

	B	S (K12)	K l
wild	wild	wild	wild
rII	mutant	wild	dead

    All three E. coli strains are important

- on B, one can select mutants
- on S, one can measure population sizes
- on K l , one can isolate rare wild type phages.  These occur via recombination or reversion

         Functional test or test of complementation is done in the following way. One infects E. coli K l directly with a mixture of the two phages under test. If one observes massive lysis, then the mutants complement and the mutants are non-allelic.

       Experimentally, E coli K l is infected directly with three viruses each of the two mutants to be tested. This  is                   comparable to making the F₁ in higher organisms.One simply asks if this combination gives rise to plaques in the cells infected. If so, complementation has occurred and the two mutants are not allelic. If no plaques, the mutants are allelic.
 

        To perform the cis-trans test, the two mutants are placed into E. coli K in both the cis and trans arrangement. In those cases in which the cis arrangement (the two mutants contained in one virus and none in the other infecting virus) gives rise to the wild type phenotype (production of plaques on E coli K l ) but the trans arrangement gives rise to the mutant phenotype, the mutants are said to lie in the same cistron.
 

    The Recombinationa l test is performed in the following way. One infects S with 3 phages of each mutant, infection occurs and the cycle of infection, take-over, cell death, release of viruses, infection occurs. Resulting virus particles are harvested and used to infect E. coli K l to see if there are any wild types.
 

Sampling the resulting progeny from the E. coli S infection is comparable to sampling the gametes arising from an F₁ in higher organisms. (Note that in the test of complementation described above, recombination can occur directly in K l to give rise to some wild type phage; however, this is a low frequency event compared to complementation [which is an all or nothing phenomenon}).
 

The test of recombination simply asks whether recombination can occur to produce a wild type gene. For example, if the mutants (represented as X's) are as drawn below:
 
 

-------------------------X-------------------

-----------X---------------------------------

                  <------------>

recombination can occur between them (in section marked with the line below) to give rise to a wild type gene. Recombination can, of course, occur outside of the designated area; however this recombination would not give rise to a wild type gene.

         In Benzer's early experiments, 8 r_II mutants were examined. In the 28 experiments in which the 8 mutants were mixed and used to infect E coli K l , 6 of the mutants were grouped into one cistron while the remaining two were grouped in a second cistron. When all 28 mixtures were first used to infect E. coli S all gave recombinant wild type phages. In other words,  mutants belonging to the same unit of function were nevertheless showing recombination.  Thus, like in the case of the lz locus in Drosophila and the wx locus in maize, recombination within a unit of function was occurring.

        Importantly, the 6 mutants falling in the same cistron mapped together and separate from the other two mutants in the other cistron. Likewise, the two mutants of the second cistron mapped together. In other words, the mutants were not intermixed: In this regard the test of functions and the test of recombination, although not giving identical results, were showing compatible or consistent results.   The data are really no different than what we saw with the lz locus last time. The unit of function is larger than the unit of recombination.