P I E R R E SCHAEFFER
I. T h e D e f i n i t i o n of T r a n s f o r m a t i o n a n d t h e F a c t s o n W h i c h I t S t a n d s 87
A. D e f i n i t i o n 87 B . P r e v i o u s R e v i e w s 88 C . D i s c o v e r y 89 D . D N A as t h e T r a n s f o r m i n g S u b s t a n c e 89
E . D N A as t h e G e n e t i c S u b s t a n c e 90 F . G e n e t i c H e t e r o g e n e i t y of D N A E x t r a c t s 96
I I . T h e Q u a n t i t a t i v e S t u d y of T r a n s f o r m a t i o n 97
A. C h o i c e of t h e S y s t e m 97 B . P r e p a r a t i o n of D N A 98 C. T i m e C o u r s e of a n E x p e r i m e n t 98
D . D o s e - R e s p o n s e C u r v e 100 I I I . T h e P r e s e n t E x t e n t of T r a n s f o r m a t i o n 100
A. T r a n s f o r m a b l e S p e c i e s 100 B . T r a n s f e r a b l e C h a r a c t e r s 101 C. N a t u r a l Occurrence of T r a n s f o r m a t i o n 101
D . Interspecific T r a n s f o r m a t i o n s 102 E . T r a n s f o r m a t i o n s O t h e r T h a n B a c t e r i a l 103
I V . T h e R e q u i r e d B a c k g r o u n d K n o w l e d g e o n D N A 104
A. S t r u c t u r e of t h e D N A M o l e c u l e 104
B . Size of t h e M o l e c u l e 104 C. B a s e C o m p o s i t i o n a n d I t s D i s t r i b u t i o n a m o n g I n d i v i d u a l D N A M o l e
c u l e s 105 D . D N A R e p l i c a t i o n 106
E . H e t e r o c a t a l y t i c A c t i v i t y of D N A 108 F . A l t e r a t i o n s of t h e D N A M o l e c u l e 108 V. A n a l y s i s of t h e T r a n s f o r m a t i o n P r o c e s s 114
A. C o m p e t e n c e of t h e R e c i p i e n t B a c t e r i a 115 B . P e n e t r a t i o n of D N A i n t o C o m p e t e n t C e l l s 119 C . S y n a p s i s of t h e I n t e r a c t i n g D N A ' s 125 D . I n t e g r a t i o n a n d G e n e t i c R e c o m b i n a t i o n 127
E . P h e n o t y p i c E x p r e s s i o n 139 V I . T r a n s f o r m a t i o n as a T o o l in G e n e t i c A n a l y s i s 140
A. G e n e t i c A n a l y s i s of B a c t e r i a l P r o p e r t i e s a n d F u n c t i o n s 141
B . G e n e t i c A n a l y s i s of P r o t e i n F i n e S t r u c t u r e 143
R e f e r e n c e s 144 I. The Definition of T r a n s f o r m a t i o n a n d the Facts on W h i c h It
A . D E F I N I T I O N
Bacterial transformation today can be denned as the integration with the genome of a recipient cell of a small piece of exogenous genetic m a t e -
* N o t e added in proof: this Chapter was written in the fall of 1962.
88 P I E R R E SCHAEFFER
rial, extracted from a donor cell and introduced into the receptor as part of a free D N A particle.
M a n y years of intensive study were necessary before this definition could be given. Several points are worth emphasizing:
1. T h e definition implies t h a t the genetic material of bacteria is identi
fied with D N A (deoxyribonucleic acid) ; this is precisely the fundamental notion introduced by the study of transformation. The fact t h a t in bacteria genetic information can also be transferred by more complex structures, such as a chromosome, an episome, or a virus (see Chapters 1, 2, and 4, respectively, this volume), makes the specification t h a t in transformation the carrier is free D N A , an essential p a r t of the definition.
2. By integration is meant some sort of permanent association between the reacting genetic materials, such t h a t the exogenous piece will be repli
cated in pace with the rest of the genome and have its genetic capabilities normally expressed in the phenotype of the transformed cell. This as
sociation is generally believed to establish itself by a substitution of the exogenous segment for its counterpart in the original genome (see Section V,D,2,e, for a discussion).
3. The contribution of the donor strain is limited to a short segment of genetic material. While the same can be said of the other bacterial recombi
nation processes, the fact is of such significance for understanding the mechanism of transformation t h a t its inclusion in the definition seems justified.
4. Two parental strains participate in the phenomenon; as their qualifi
cation as donor and receptor illustrates, the roles they play are essentially different. But in principle at least, they can be freely interchanged, the phenomenon requiring no sexual differentiation of the reacting strains.
5. Although new genetic factors m a y be created in the process of trans
formation, they arise by recombination between preexisting genetic struc
tures, a mechanism incompatible with transformation being referred to as a mutation.
Those studies on transformation t h a t permitted the above definition to be arrived at will be reviewed in this section.
B. PREVIOUS R E V I E W S
A landmark in the development of bacterial transformation and molecu
lar biology, the classical paper of Avery et al.,1 m a y be quoted as the first review on the subject; the most complete and up-to-date is by R a v i n .2 T h e successive advances and the various points of view m a y be followed along the years with the reviews of M c C a r t y ,3 Austrian,4 Hotchkiss,5' 6 E p h - r u s s i - T a y l o r ,7 , 8 Z a m e n h o f ,9'1 0 and T h o m a s .1 1
T h e phenomenon was discovered by Griffith,1 2 as he was studying im
munity to pneumococcal infections. While mice will die in two days of bacteriema, when injected subcutaneously with a few encapsulated, smooth pneumococci, they will survive infection with enormous numbers of nonencapsulated, rough mutants. B u t if the latter are injected together with heat-killed smooth organisms (the "vaccine"), a fatal infection again develops, with the invading pneumococcus being encapsulated and of the same serological type as t h e vaccine. I t was then believed t h a t the t y p e - specific capsular polysaccharide of the vaccine was responsible for the observed transformation of the rough inoculum. T h a t this was not so was recognized by Sia and D a w s o n ,1 3 who were able to obtain transformation in in vitro cultures.1 4 T h e preparation of heat-stable, cell-free transforming agent was described by Alloway.1 5
D . D N A AS THE TRANSFORMING SUBSTANCE
With the active "principle" available in solution, its chemical identifi
cation was achieved by Avery and his collaborators. I t was observed t h a t trace amounts of the transforming agent (1 m/xg./ml.) produced t r a n s formed bacteria, but t h a t any desired amount of it could subsequently be extracted from a transformed clone : obviously the agent was being synthe
sized by the transformed cells, which had therefore simultaneously acquired two new biosynthetic abilities, one leading to the capsular polysaccharide, the other to the transforming substance, both abilities being regularly transmitted to the progeny.1
T h e transforming agent was shown to be D N A . A wealth of arguments was produced in support of this conclusion: chemical, physical, enzymic, and serological tests all agreed in indicating t h a t activity is associated with D N A alone. Among the best pieces of evidence was the fact t h a t crystal
line proteases and R N a s e (ribonuclease) leave the activity unimpaired, while traces of D N a s e (deoxyribonuclease), whatever its origin, destroy it readily. Crystalline D N a s e was not available at t h a t time, b u t tests made l a t e r1 6 - 1 8 confirmed the results entirely. Overwhelming as it was, the evidence still met with skepticism,1 9 the reasons for which are instruc
tive to consider briefly.
At least half a dozen capsular transformation reactions were already k n o w n ,2 0 which m e a n t t h a t the transforming substance had to exist in a large number of specific forms. As the progress of enzymology and immu
nology had already demonstrated, proteins did display such specificity and therefore could conceivably be the transforming substance, while D N A , still believed to be a monotonous polymer of a tetranucleotide u n i t ,2 1
90 P I E R R E S C H A E F F E R
was considered to be lacking in specificity. Considering the primitive state of knowledge of D N A composition, Avery's conclusion came too early to make sense. Hotchkiss spent much effort in reducing the maximal protein content of the transforming substance to 0.02%2 2 and eventually proposed t h a t anyone who again suggested proteins as the active factor of transform
ing extracts would have to buttress the argument with new experimental evidence. The challenge was not taken up, and did not need to be, since meanwhile the supposedly uniform composition of D N A was disproved by Chargaff.2 1 Polysaccharides could no longer be suspected of activity, when transformation reactions were extended to characters in the expression of which they were not involved.2 3 E a r l y additional reasons for identifying the "transforming principle" with D N A deal with resistance to inactiva- tion by physical a g e n t s ,1 0'1 7 purification by electrophoresis1 , 2 4 and elec
tron microscopy.2 2
E. D N A AS THE GENETIC SUBSTANCE
The meaning of Avery's discovery was at first far from obvious. The notion slowly emerged t h a t D N A is the genetic substance, and t h a t recom
bination can occur between the genome of a living bacterium and the dis
solved genome from a dead one. For the sake of clarity and at the risk of disregarding their chronology, the facts which contributed to the elabora
tion of this notion will now be presented under three headings.
1. RECIPROCAL TRANSFORMATIONS
From smooth strains of pneumococcus, rough m u t a n t s are isolated, which in turn produce a second-step m u t a n t , with an "extreme rough" colonial appearance. Like its parental rough form, the latter is unable to produce a n y capsular substance. With the symbols originally in use provisionally adopted, the three strains are referred to as S , R, and E R , respectively. The following reactions were shown by T a y l o r2 5 to occur*:
E R + (R) - * R (1) R + ( E R ) -> E R (2)
The possibility of a reciprocal transformation reaction was thereby es
tablished. T h a t this reciprocality was the rule rather t h a n the exception was later demonstrated by studies on other systems: Sb ^ Sd , between the serological types b and d, in smooth strains of Hemophilus influenzae™;
Sm1 ^ Sui2 , between type I I I m u t a n t s of pneumococcus, producing
* I n d e s c r i b i n g t r a n s f o r m a t i o n r e a c t i o n s , t h e f o l l o w i n g c o n v e n t i o n is a d o p t e d : the r e c i p i e n t s t r a i n is w r i t t e n first, t h e d o n o r s e c o n d , p a r e n t h e s e s i n d i c a t i n g D N A p r e p a r a t i o n ; t h e d e s i g n a t i o n of t h e t r a n s f o r m a n t s o b t a i n e d f o l l o w s a n arrow.
various subnormal amounts of capsular material2 7; Sp+ ^ Sp~, between sporogenous and asporogenous forms of Bacillus subtilis28>29 etc.
Whenever the reciprocality of transformation cannot be demonstrated, the difficulty is one of selection only. Any Xs ;=± Xr system, between the sensitive and the resistant forms to an antibacterial agent X , can be taken as an illustration: the only transformants difficult to select for are the sensitive ones. B u t even in such cases, t h e reciprocality of transformation can be demonstrated, as shown b y Hotchkiss and M a r m u r ,3 0 if a selectable marker, linked to the X marker, is available*:
Mtl~Smr X (Mtl+Sm8) -> selected Mtl+ transformants, 20 % of which are also Sm8.
The same demonstration was also given by Goodgal3 1 in Hemophilus, where the Sm and Cm (cathomycin) markers are linked:
Sm8 Cmr X (Smr Cms) —» selected Smr transformants, m a n y of which are also Cm8.
Reciprocality therefore is the rule and excludes the possibility t h a t one of the strains might have lost a determinant present in the other.2 5 T h e following interpretation was suggested : different forms of a gene are pres
ent in the two strains, each strain having one, and transformation consists in an exchange reaction of the allelic form present in the recipient genome for the one present in the exogenous D N A (MacLeod and K r a u s s3 2; T a y l o r2 5) .
2. INDEPENDENT TRANSMISSION OF CHARACTERS
I n Avery's laboratory, capsular transformation only was tried, and with this one marker being followed, the transformed cells appeared iden
tical with the donor cells. A demonstration t h a t this was not the case can be found in Langvad-Nielsen's w o r k .3 3 Still following Griffith's procedure of transformation in vivo by heat-killed "vaccine," this author was concerned with the sterility of the vaccine, and for this reason differentiated the in
teracting strains with a sulfonamide (Sf) marker. His results can be sum
marized in the following way :
R Sf X (SIT Sf) -> Su Sf (3)
R Sf χ (S„ Sf) -+ S„ Sf (4)
With phagocytosis in the mouse selecting for smooth transformants, it can be seen t h a t the latter retained the Sf marker present in the receptor:
they differed from both parental strains, having acquired only one of the two markers present in the D N A and being in fact recombinants. (Double transformants, identical with the donor, were presumably also present, but
* Mtl for g r o w t h b e h a v i o r o n m a n n i t o l , Sm for b e h a v i o r in s t r e p t o m y c i n ; t h e t w o c h a r a c t e r s are l i n k e d i n p n e u m o c o c c u s .3 0
92 P I E R R E S C H A E F F E R
being rare were overlooked.) While the recombinational nature of transfor
mation had been demonstrated, the author only concluded, too modestly, t h a t the occurrence of transformation had been confirmed, and his demon
stration went unnoticed for some time.
The next illustration of the one-by-one transmission of characters is found in the stepwise transformation of an E R into an S strain (Taylor3 4;.
A description of the relations existing between these two strains must first be given. The peculiar colonial appearance of E R m u t a n t s results from both their inability to produce any capsular substance and their tendency to grow in the form of long filaments (Austrian3 5). These two characters, capsule production (S+) and filament formation (Fil+), were shown by Austrian to m u t a t e independently.3 6 Thus, an ordinary smooth strain of serological type I I I is Fil" Stn , a rough m u t a n t derived from it is Fil~ STn , and an extreme rough m u t a n t , isolated from the latter, Fil+ STn . (The Fil character only was changed in the reactions 1 and 2 described above).
Now the two following reactions were obtained in succession by Taylor3 4 :
Fil+ Sli X (Fil- Sin) -> FUSIT (5) Fil- Sjτ (from r e a c t i o n 5) X (Fil' Shi) -> Fil~ Sin (6)
Here is a case where one and the same D N A preparation is shown to contain two distinct genetic determinants (Fil~ and Sfu) which, in trans
formation, are transmitted singly. The same is shown also in Austrian and MacLeod's case: the ability of pneumococcus to produce a somatic, type- specific protein and a capsular polysaccharide can be independently lost by mutation and acquired by transformation.2 3-3 7
A clear formulation, however, t h a t characters are, as a rule, transferred independently, was only given by Hotchkiss,3 8 studying simultaneous transformation for both encapsulation and penicillin resistance (Penr)y i.e., two physiologically unrelated markers, of which the latter is easily selected and quantitatively recovered. I n the reaction: S~ Pen* X (S+ Penr), trans
formation occurred for both markers, but most individual transformants had acquired one of them only; a very few doubly transformed clones could be detected in this case.
Another important point was made. A high level of penicillin resistance, obtained by selection of successive mutations, had been attained in some strains. When the sensitive wild type was given D N A from such a strain, it acquired, by transformation, the increments in resistance one by one, just as it had by mutation (although not necessarily in the same order).
The point here is t h a t with D N A so similar in all its potentialities with the genome of the organism from which it was extracted, another powerful argument is given in favor of the view t h a t D N A is the genome itself in solution.3 8
3. HETEROALLELIC TRANSFORMING AGENTS
Still other arguments supporting the above conclusion had in fact al
ready been produced in studies following quite a different line: the trans
formation analysis of m u t a n t s producing reduced amounts of an otherwise normal capsular polysaccharide, and appearing thereby as intermediary between the rough (S~) and the normal smooth (SN+) conditions.
With such a n intermediary m u t a n t of type I I (Sni), MacLeod and Krauss3 2 observed the following reactions:
SJr X (8ht) - > Stn (7)
SJi X (SilN) - ShN (8)
Shi X (SÎIN) -» Stiy (9)
T h e results were those to be expected, if the two D N A ' s employed were carrying distinct allelic forms of a gene for type I I polysaccharide synthesis, t h e third and extreme form of which would be present in t h e rough strain.
Under t h e assumptions made, transformation again would consist in an exchange reaction.
A similar situation in type I I I was exploited further by Ephrussi-Tay-
lor.3 4 > 3 9-4 0 For the sake of simplicity, only two of her intermediary m u t a n t s
will be mentioned,
•Here again, stepwise intratype trans
formation was observed, as appears in the following reactions:
SJi X (StiiJ -> Sim ( e x c l u s i v e l y ) ( 1 0 )
Sli X (Sni2) —• Sin2 ( e x c l u s i v e l y ) ( 1 1 ) SÎrii [from r e a c t i o n (10)] X (Sui2) —> Sni2
( 1 2 ) [not e x c l u s i v e l y , h o w e v e r ; see r e a c t i o n (12a)]
Several important additional observations were made.
a. Loss of the Replaced Segment. Transformants in reaction (12) have at the same time acquired the
Stn2 determinant and lost t h e
Sti^factor they posessed originally. This was shown in the reaction:
Sli X [SÎn2 , a t r a n s f o r m a n t f r o m r e a c t i o n (12)] —-> Stn2 ( e x c l u s i v e l y ) (13)
(Had Stiii transformants been formed, they would have been detected.) Replacement of one determinant by another is directly demonstrated here, together, incidentally, with the haploidy of the organism. T h a t the chromo
somal marker, allelic to the one introduced b y transformation, is not only displaced, b u t actually lost at integration, has been confirmed by m a n y a u t h o r s .3 1-4 1"4 3
b. Test for Allelism. While the
Stn2 agents are mutually ex
clusive, this is not true of other pairs of determinants, like Fil" and Stn ,
94 P I E R R E S C H A E F F E R
t h e coexistence of which has been demonstrated [reactions (5) and (6)].
T h e reason for this difference in behavior was assumed to be an allelic relation existing between the members of the former pair only, an assump- tion supported b y t h e similar function and t h e common gene of origin of the two intermediary Sfn genes. A new kind of test for allelism was thereby provided, ample use of which was made later.
c. Allogenic Transformations. Reaction (12), as written, is incomplete, since in addition to the
Stn2 transformants mentioned, another class of (rare) transformants,
(Nfor normal), is formed. T h e correct reaction is therefore:
S Ï J / I X (Sin2) SÎn2 + SniN (12a)
I t shows t h a t more t h a n one kind of reaction is possible, when a given pair of genomes is reacting. T h e
Stn2 transformants, with their acquired char- acter already present in the donor, are said to result from an "autogenic"
reaction, and are expected; b u t the
SfnN transformants unexpectedly dis- play a character, absent from both parents, which must have arisen b y intragenic r e c o m b i n a t i o n .3 9 , 4 0 Such transformants, said to be "allogenic,"
are detected from the normal-looking smoothness of their colonies. Having arisen by recombination, their genotype might be either
Sti^ Stu2 (with the simultaneous presence of the two mutated determinants leading, by summation of their synthetic abilities, to a normal polysaccharide produc- tion) , or
StnN (with elimination of both mutated sites and reconstitution of a wild capsular gene). T h a t the latter event alone is responsible for the formation of the normal smooth transformants is shown by the following reaction :
Su X [SinN , f r o m r e a c t i o n (12a)] —> SiiiN ( e x c l u s i v e l y ) (13)
Stm Stn2 recombinants, therefore, if they are formed, do not con- tribute to the normal smooth class, the defects, rather t h a n t h e abilities, of their determinants being additive. The following interpretation was p r o p o s e d3 9 , 4 0 to account for the creation, by transformation, of genetic factors absent from both " p a r e n t a l " strains.
One linear genetic region exists in t y p e I I I pneumococcus, responsible for its specific encapsulation. A t different sites along this region, mutation is possible, the various capsular m u t a n t s observed corresponding to various mutational sites. Essentially a recombination between two genetic struc- tures, transformation requires, when carried out with two heteroallelic mu- tants, t h a t their mutated sites do not overlap exactly. Transformation m a y be of two types, autogenic and allogenic, both types of transformants arising by replacement of one segment of the host's genome by another,
carried in with the D N A . As illustrated by Fig. 1, replacement implies the occurrence of two events, formally analogous to crossing over. The capsular reactions studied imply t h a t the site homologous to the one m u t a t e d in the bacterium always be included in the replaced segment; whether a t r a n s formation turns out to be autogenic or allogenic depends simply on whether the location of the second "crossing-over" is such t h a t the m u t a t e d site of the donor is included in, or excluded from, the replaced s e g m e n t .3 9 , 4 0
molecule oooooooooooooo ο
F I G . 1. A u t o g e n i c a n d a l l o g e n i c t r a n s f o r m a t i o n s (after E p h r u s s i - T a y l o r3 9'4 0) . T h e t r a n s f o r m i n g m o l e c u l e , m u t a t e d a t s i t e 2, is s h o w n s y n a p s e d w i t h t h e h o m o l o g o u s region, m u t a t e d at s i t e 1, of t h e c a p s u l a r g e n o m e of t h e cell. Ο Ο Ο , a u t o g e n i c t r a n s f o r m a t i o n ; , a l l o g e n i c t r a n s f o r m a t i o n .
4. EVIDENCE FROM OTHER F I E L D S
The notion t h a t the genetic substance is D N A , established by the study of bacterial transformation, was soon found to fit admirably a large number of observations (cf. réf. 6 for a review), some of which will now be mentioned.
a. I n higher organisms D N A , like genes, is confined to chromosomes6 , 4 4 ; with due recognition of the peculiarities of the bacterial "nucleus",4 5 this is also true of b a c t e r i a .4 6 , 4 7
b. Variable with the species, the amount of D N A per cell within a species is constant, but follows the degree of ploidy whenever the latter varies, as is the case in gametogenesis.4 8
c. D N A alone is endowed with the metabolic stability t h a t is to be ex
pected of the genetic substance6; its genetic (transforming) activity was found to be independent of whether the physiological function under its control is expressed in the cell or n o t .4 9
d. A number of agents altering the structure of D N A are powerful m u t a gens at the same time: ultraviolet (UV) light, mustard gas, acridine dyes
(see ref. 6 ) , h e a t ,5 0 nitrous acid,5 1 base analogs, etc.
96 P I E R R E S C H A E F F E R
e. Recent biochemical studies have come very close to demonstrating directly t h a t D N A is endowed with both the autocatalytic and the specific heterocatalytic activities, upon which the definition of the gene is based.
D N A is required as a primer, and most likely as a specific template, for its own enzymic synthesis.5 2 , 5 3 I t is also required as a specific template for the synthesis of enzymes, or more precisely, of the messenger R N A ' s which confer their specificity to the proteins being synthesized in the cytoplasm
(see Chapter 8 ) .
f. T h e situation in viruses will break the monotony of this accumulation of unfailingly concording data. Viruses differ from cells in having one kind of nucleic acids only, D N A or R N A as the case m a y be, wrapped in a pro
tective coat of specific viral proteins. Whatever its nature, however, the nucleic acid alone is infectious; once introduced into the host cell, it is able by itself to give rise to mature, infective p a r t i c l e s .5 4 - 5 6 While the secondary role played by proteins is thereby confirmed, the results demon
strate t h a t R N A m a y in some cases be the primary source of genetic speci
ficity. Although this apparent oddity m a y have some bearing upon the cel
lular origin of some viruses, it casts no more doubt on the genetic nature of D N A than do the known cases of extrachromosomal heredity on the exist
ence of Mendelian heredity.
F . GENETIC HETEROGENEITY OF D N A EXTRACTS
D N A extracts from a single clone contain more t h a n one genetic factor, and these are as a rule transmitted singly. I n Bacillus subtilis transforma
tion, for instance, just as m a n y transforming activities can be detected in wild-type D N A as auxotrophic markers have been introduced in the re
cipient strains: the entire genome of the donor organism is present in solution in the extract. T h e rule of unit factor transmission still generally applies.
If to every bacterial character, whether recognized or not, there corre
sponds one specific gene, thousands of genes must be present in the extract, either as specific segments along one linear D N A structure, or as discrete D N A particles, each containing one gene or more. Indeed, it is now known t h a t a conventional D N A extract contains some fifty molecular species, with probably a few tens of genes accommodated in each of them. Suffice it here to point out (1) t h a t a gene (and a fortiori a marker, which m a y be a mere mutated site in a gene) is smaller t h a n the usual D N A particle, and (2) t h a t the heterogeneity of D N A particles in extracts, first deduced from genetic considerations, prompted chemical and physical studies which fully confirmed it.
With the definition of transformation now fully justified, particular as
pects of the phenomenon will be described.
II. The Q u a n t i t a t i v e S t u d y of T r a n s f o r m a t i o n
I n the experience of early workers, transformation was a rare and poorly reproducible phenomenon, affecting a fraction of the population t h a t was hard to estimate, but very small. The main difficulty was with competence, a property to be dealt with in another section. B u t the choice of the recipi
ent strain, of the character transferred, the quality of the D N A prepara
tion, and the time course of the reaction also are of importance and will now be considered.
A. CHOICE OF THE SYSTEM
Not all strains of a species can be transformed. Unlike competence, a physiological state, transformability is a genetically determined property.
I n pneumococcus, the amount of capsular substance t h a t a strain is able to produce seems to bear an inverse relation to its transformability.2 7 Avery et al. reported t h a t the rough strain R36 was exceptional, among a number of rough clones, in being highly transformable1; so are the strain R d of H. influenzae?1,58 some strains of s t r e p t o c o c c i ,5 9 , 6 0 the m u t a n t $ 1 6 8 of B. subtilis M a r b u r g ,6 1 the rough strain C of Rhizobium lupini,62 the strain Ne-11 of Neisseria catarrhalis.™ N o ways are known of selecting for highly transformable clones, a rare transformant from a poorly transformable strain retaining the original low level of transformability. Since the search for good transformable strains must be conducted blindly, and in general brings poor rewards, it is not surprising t h a t a few selected strains are being used the world over in studies on transformation. Since serial transfers of a transformable strain m a y lead to a loss of transformability, early lyo- philization of such strains is recommended.
T h e choice of the character to be transferred m a y be dictated by the sub- ject under study (see Section VI) ; as a rule, preference will be given to characters easily selected for, permitting a quantitative recovery of the transformants.3 8 I n t h a t respect, capsular characters are well known as unsuitable,6 4 , 6 5 their expression apparently requiring a restricted disper
sion of the cells,6 6 usually obtained by adding an antiserum. B u t transfor
m a n t s are trapped in the aggregates, and in some cases the antiserum m a y even be inhibitory to the desired reaction.6 6 Anti-R antibodies are not re
quired for the appearance of competence, and can be dispensed with in the study of transformations other t h a n capsular o n e s .3 8 , 6 6 Even with an ac
curate count of the transformants, transformation frequencies are not the same for all characters, the probability of integration being an innate prop
erty t h a t varies with each marker.
98 P I E R R E SCHAEFFER B . PREPARATION OF D N A
Although transformation m a y be obtained with D N A directly liberated from the donor in the presence of the receptor c e l l s ,3 8 , 6 7 quantitative work demands t h a t a purified extract of known D N A content be used. The classi
cal method of pneumococcal D N A preparation, involving deoxycholate- induced lysis of the cells, deproteinization by chloroform, and ethanol pre
cipitation, has been described and improved m a n y t i m e s .1 5 , 3 2 , 3 9 , 6 8 , 6 9 R N A is usually removed by R N a s e treatment, but removal of the inert polysac
charides is today seldom carried out. Deproteinization, a tedious step re
quiring m a n y treatments when chloroform is used, is obtained more rapidly with detergents, or by phenol t r e a t m e n t .6 1 , 7 0 Direct phenol extraction of D N A7 1 has also been used in transformation s t u d i e s .7 2 , 7 3
With other bacterial species, the classical method is generally applicable only after the D N A has been solubilized, cell lysis requiring special treat
m e n t s .7 0 Mechanical or sonic disruption of the cells should be avoided;
even pipetting of the solutions should be performed as gently as possible and kept to a minimum (see Section IV,F,2).
Sterilization of the extract is most simply obtained by the last ethanol precipitation (filtration should be avoided). This procedure is inadequate, however, with D N A t h a t contains spores. I n the case of spore-formers, D N A extraction should be made only from cultures growing exponentially in a rich medium, or, when possible, from asporogenous mutants. Contami
nated preparations can be rid of spores, however, by inducing germina
tion with alanine and glucose before alcohol treatment, or by killing the spores directly with phenol.7 4
Stock solutions of D N A will keep for years in saline, or better sometimes in 2 M N a C l .7 5 Since molecular aggregation occurs and increases with a g e ,6 9 , 7 6 applying a chloroform treatment to old solutions before use m a y be good practice.6 9 Titration of D N A m a y be chemical,7 7 spectrophometric, or b i o l o g i c a l .6 5 , 7 8
C. T I M E COURSE OF AN EXPERIMENT
The general principles upon which an experiment is to be scheduled, laid down by H o t c h k i s s ,6 5 , 7 8 can be summarized as follows. The number of transformants become meaningful only when expressed relative to the number of viable cells exposed to t r e a t m e n t ; detemination of the ratio (i.e., the frequency of transformation) requires two bacterial counts. An accu
rate count of the transformants demands not only t h a t they be quantita
tively selected for, but also t h a t their progeny cannot disperse before selec
tion is applied; this can be insured either by timely plating on a solid medium, or by adding the proper agglutinins to a liquid o n e .6 5 An accurate count of the viable population during treatment requires: (a) a sharp end of the exposure to D N A (terminated by treatment with D N a s e ) , and (6) a
treatment t h a t is short, relative to the generation time of the organism.
With exposure thus necessarily short, use of a highly competent culture is all the more important.
How these principles apply in actual practice partly depends on the system being used. T h e cultural conditions have first to be empirically adjusted, as described in Section V,A,1. T h e time a t which competence is maximal is then determined in kinetic experiments of the kind described by H o t c h k i s s2 8 7 and by T h o m a s .7 9 Needless to say, reproducible kinetics will not be obtained until all cultural conditions are strictly standardized.
D N A then can be added, at a precisely known time, and D N a s e 5-10 minutes later. W h a t is next to be done depends on the character being transferred. With the acquisition of a resistance to a bactericidal drug, like streptomycin, exposure to the drug will have to be postponed until resist
ance is expressed, otherwise even the transformants will be killed.8 0 Time alone is of no avail, however, when nutrients for syntheses are lacking; with competence often appearing at the end of exponential growth, in a medium approaching exhaustion, it is good practice to dilute the culture with fresh medium before incubating for expression.8 1 If the diluted culture is plated at various time intervals directly onto a drug-containing agar medium
("immediate challenge"), an expression curve is obtained, giving the num
ber of transformants expressed at the time of plating (Fig. 2, curve A) .5>7 9
F I G . 2. E x p r e s s i o n of r e s i s t a n c e t o s t r e p t o m y c i n a n d g r o w t h of t h e t r a n s f o r m a n t s (after E p h r u s s i - T a y l o r8 2) . D N a s e is a d d e d at t0 , t h e t i m e zero of e x p r e s s i o n (the d u r a t i o n of c o n t a c t w i t h D N A , ca. 5 m i n u t e s , is n e g l i g i b l e ) . T h e p r o c e d u r e s f o l l o w e d for o b t a i n i n g c u r v e s A a n d Β are d e s c r i b e d i n t h e t e x t . I n a c o m p l e t e m e d i u m , a t 3 7 ° C , 60 t o 75 m i n u t e s are r e q u i r e d for c o m p l e t e e x p r e s s i o n .
100 P I E R R E SCHAEFFER
Alternatively, the DNase-treated culture m a y at once be incorporated into a plain agar medium and incubated for 2 hours, a drug-containing agar layer being then poured on top. This second procedure ("belated chal
lenge") simply gives the total number of transformants.6 5 The two proce
dures m a y also be combined into a third, whereby samples of the treated liquid culture, taken out a t various times after addition of D N a s e , are sub
mitted to a belated challenge.8 2 This leads to curve Β of Fig. 2, representing the multiplication of the transformed cells; the two curves superimpose when expression is completed.8 2
When the drug does not kill the sensitive cells a t once, as is the case with sulfonamides, or when a new biosynthetic ability has been acquired by transformation, immediate challenge can be used after D N a s e addition, since expression takes place during residual growth on the plate. Other se
lective methods m a y have to be devised for special cases, like acquisition of motility, ability to sporulate, etc.
Fox and H o t c h k i s s8 3 have introduced a very useful modification of the standard procedure just described, which consists in storing at deep-freeze temperatures aliquots of a competent culture to which 10-15% glycerol has been added.
D . D O S E - R E S P O N S E CURVE
When the transformants counted are expressed as a function of D N A concentration, a saturation curve is obtained similar to the one in Fig.
3 5 8 , 6 4 , 6 5 , 7 9 shows a linear part, at low D N A concentrations, and a hori
zontal p a r t at higher, saturating ones. Minor deviations from the curve have occasionally been observed in the plateau region.7 6
10 100 /y 1 0 0 0 DNA, m/xg/mi
F I G . 3 . T r a n s f o r m a t i o n as a f u n c t i o n of D N A c o n c e n t r a t i o n .
The linear part, extrapolated down to zero concentration, goes through the origin, as expected if one D N A particle is enough to transform one bac
terium. This important point has been stressed by m a n y a u t h o r s .6 4 , 6 5 > 7 9 , 8 4
Formally, the treatment of a bimolecular reaction can be applied to the re
action of a bacterium with D N A .7 9 , 8 3 , 8 5 - 8 8
T h e proportional response p a r t of the curve lends itself to, and should be exclusively used in, biological titration of transforming D N A , the concen
tration of which is measured by the slope of the linear p a r t ; information of another, qualitative sort is obtained by studying the plateau r e g i o n .6 5 , 7 8 III. The Present Extent o f T r a n s f o r m a t i o n
A. TRANSFORMABLE SPECIES
T h a t ability to transform is not a unique attribute of the pneumococcus first became apparent with the discovery by Alexander et al. of transfor
mation in Hemophilus89 and Neisseria.90 Since then, transformation has been reported to occur in a number of bacterial species (bibliography in R a v i n2; see also J a r a i ,9 1 Wacker and Laschet,9 2 Perry and S l a d e ,9 3 who recently reported on transformation in streptomyces, Escherichia coli, and streptococci, respectively).
A new transformable species nowadays raises interest mostly as a m a t e rial making some new bacterial function accessible to genetic analysis
(see Section V I ) . Bacillus subtilis M a r b u r g deserves special mention in this respect. This strain can be grown and transformed in chemically de
fined m e d i a ,6 1 , 7 5 , 9 4 and with the practically unlimited number of genetic markers t h a t can be obtained, some precise genetic mapping has already been d o n e .9 5 - 9 7 I n addition, the organism produces spores, pigments, fla
gella, mesosomes,9 8 extracellular enzymes, a t least one antibiotic,7 4 and probably bacteriocins ; moreover, phages,9 9 some of them t r a n s d u c i n g ,1 0 0 - 1 0 3
are known in this strain, which itself carries an inducible prophage.7 4 B . TRANSFERABLE CHARACTERS
I n transformable strains, all characters t h a t can be recognized can also be transferred by D N A (an enumeration of specific examples m a y be found in ref. 2 ) . This m a y mean either t h a t cytoplasmic mutations do not occur in bacteria, or t h a t most of them are lethal.
However, two preliminary reports have recently appeared, claiming t h a t stable genetic transformation had been induced by R N A . I n one of them, the ability to produce a constitutive penicillinase was transferred from a penicillin-resistant m u t a n t to a sensitive strain of B. subtilis10*;
in the other, a pyrimidineless m u t a n t of Neurospora was said to be restored to prototrophy by R N A .1 0 5 I n both cases, R N a s e inactivated the extracts, while D N a s e had no action. Independent confirmation of these unexpected results is highly desirable.
102 P I E R R E SCHAEFFER
A potentiation of DNA-induced transformation by an R N A fraction
"closely associated with D N A " has been described by Spizizen in B.
subtilis61*106 ; it seems, however, to be detectable only when special markers are being t r a n s f e r r e d .7 5'1 0 6 No genetic effect was attributed in this case to R N A , which was believed to help with the expression, and therefore the scoring, of the transformants. If any one of these specific activities of R N A is confirmed, a more detailed characterization of the active R N A will be needed, since the same activity would not be expected for messenger R N A1 0 7 and for D N A - R N A h y b r i d s ,1 0 8 particularly with respect to genetic effects.
C. NATURAL OCCURRENCE OF TRANSFORMATION
Since transformation in the laboratory requires the preparation of cell extracts, the question arises of its natural occurrence.1 0 9 Penicillin l y s i s3 8 need not be invoked as a way of liberating genetically active D N A in nature, since this liberation takes place spontaneously, either by autoly
s i s1 1 0 , 1 1 1 or even by a nonspecified mechanism, during exponential g r o w t h6 7; the fact is t h a t transformation does occur in untreated mixed cultures.6 7 Transformation m a y well occur in nature, b u t its actual con
tribution to gene flow is hard to evaluate.
D . INTERSPECIFIC TRANSFORMATIONS
Transformation is not a strictly intraspecific phenomenon. Leidy et al.112 and S c h a e f f e r8 1 , 1 1 3 simultaneously observed interspecific transformations in Hemophilus. The first mention of their occurrence, however, is found in papers by Balassa, working with Rhizobium.114"115 These reactions were later found to be widespread, occurring between pneumococcus and s t r e p t o c o c c u s5 9 , 1 1 6 and among species of Neissena6S>117 and Bacil
lus.11* Cases of interspecific recombination by transduction or conjugation are also steadily increasing. Genetic compatibility being one of the best criteria upon which species are defined, a classification of bacteria which took into account the recent genetic evidence would end up with a smaller number of highly polytypic s p e c i e s1 1 9 , 1 2 0 (see also Chapter 9).
Interspecific recombination, as a rule, is rare, when compared with the corresponding intraspecific reaction; the suggestion has been made t h a t transformation frequencies might reflect the degree of kinship existing be
tween the reacting s t r a i n s .8 1 , 1 1 2 Exceptions to the rule have been observed, indicating t h a t genetic factors other t h a n kinship m a y affect transforma
tion f r e q u e n c i e s .5 9'1 2 1 Genetic factors influencing these frequencies are known to exist, even in intraspecific t r a n s f o r m a t i o n .1 1 2 , 1 2 2
The meaning of the low frequencies usually obtained in interspecific transformation reactions and the way in which these reactions can be used to recognize steps in the transformation p r o c e s s1 2 1 will be discussed in a later section.
E . TRANSFORMATIONS OTHER T H A N BACTERIAL
Transformation has not yet been found in any microbial group other than bacteria. A good deal of effort has been spent trying to detect its occurrence in genetically well-known microorganisms, like Neurospora, Pénicillium, and yeast, with only failure as the result. I t is not known to the writer whether Cyanophyceae have been tried.
Infection of cells by viral D N A is outside the scope of this review, ex
cept in cases where acquisition by the cell of a stable new trait, rather than cell death, results from the infection. D N A from a temperate phage can lysogenize an infected bacterium; and with special transducing phages as the D N A source, this lysogenization, leading to the integration of bacterial genes, complies with the definition of transformation.7 3 I t seems quite plausible t h a t lysogenization and immunity can also be obtained with D N A from a lysogenic bacterium ("prophage D N A " ) ; the only data pertaining to this p o i n t1 2 3 do not seem very convincing.
On condition t h a t free viral D N A can enter the host cell, viruses them
selves, as genetic entities, are transformable during their vegetative stage.
This has only recently been established ( K a i s e r1 2 4) , and had long been the accepted interpretation of the well-known Berry-Dedrick phenomenon, in which an animal virus is "transformed" into another, serologically related o n e .1 2 5 - 1 2 8 Twenty-five years after the initial observations, how
ever, not only is good characterization of the active material as free D N A still lacking, but the possibility for a heated virus to be reactivated by a mechanism other than genetic has been established.1 2 8 1 1
Reaction of D N A with bacteria can be demonstrated at the physical, the physiological, and the genetic level by measuring uptake of a labeled D N A , synthesis of a specific enzyme or product, and recombination, re
spectively. With bacterial transformation serving as a model, reaction of D N A with animal cells begins to be investigated. U p t a k e has been dem
onstrated by using D N A ' s labeled with tritiated t h y m i d i n e ,1 2 9 - 1 3 2 radio- p h o s p h o r u s ,1 3 3 or acridine o r a n g e .1 3 0 Addition of unlabeled D N A in excess or of D N a s e being inhibitory, the label must be incorporated as a polymer
ized m o l e c u l e1 2 9 , 1 3 3; indeed, it is found in the D N A fraction of the c e l l s1 2 9 and m a y be localized in the nucleus by a u t o r a d i o g r a p h y .1 3 0 - 1 3 2 With some cells, uptake requires t h a t DNA-protein particles be made for the cells to phagocytize.1 3 0
104 P I E R R E SCHAEFFER
T h e physiological and genetic demonstrations, requiring genetic markers, are much more difficult to obtain ; b u t K r a u s was recently able to show t h a t the ability to produce t h e βΑ polypeptidic chain of hemoglobin, absent from the bone marrow cells of a patient with sickle cell anemia, was con
ferred on these cells in vitro by D N A from the bone marrow of a m a n homozygous for hemoglobin A .1 3 2 T h e physiological evidence seems thereby provided. Experiments providing genetic evidence, which would require active multiplication of the animal cells into clones and a marker lending itself to selection, have not yet been feasible (see Section V,E, however).
I V . The R e q u i r e d B a c k g r o u n d K n o w l e d g e on D N A
As a result of the identification of D N A as the bearer of genetic informa
tion, research on its physical, chemical, and biological properties has lately been progressing a t an ever-increasing pace. Much of the newly ac
cumulated knowledge is essential for an understanding of the processes involved in transformation. While no attempt is made to cover these di
vergent fields (some aspects of which are developed in Chapters 7 to 9 ) , the minimal relevant information will now be given.
A. STRUCTURE OF THE D N A MOLECULE
T h e structure of D N A proposed by Watson and Crick in 1 9 5 31 3 6'1 3 7 (see Chapter 7 ) , which integrated existing chemical2 1 and p h y s i c a l1 3 4'1 3 5 evi
dence into an illuminating model, has ever since been recognized as the one t h a t fits best all facts. This structure has been observed in all prepara
tions of native D N A , whether deproteinized or not, and even in situ in cells.1 3 8 Suffice it here to recall t h a t two levels of structure are considered.
The primary structure is t h a t of the chains or strands of nucleotides. T h e secondary structure results from the molecule being double-stranded, hydrogen bonds maintaining together specific pairs of nucleotides.
B . SIZE OF THE MOLECULE
T h e question of the size of the D N A molecule is complex and has been
"solved" only recently. T h e D N A preparations obtained by the usual methods have molecular weights of the order of 1 Χ 107 (see 140, 141 for references; this is a mean value, D N A being polydisperse). Since the amount of D N A per bacterial cell is close to 2 Χ 1 0- 1 5 g . ,1 7 , 1 4 2 or 1 X 2Q-15 g pe r n Uc l e u s ,1 4 3 it follows t h a t some 50 molecules are liberated per nucleus upon cell lysis (a figure leading to 20 genes per molecule, if there are 1000 genes in a bacterial genome).
T h e question then arises of whether these "molecules" are preexisting in the chromosome (in which case "linkers" are required to connect them
together), or are artifacts, being produced, a t the time of cell lysis, from a continuous double-stranded structure. The assumed difficulties of un
winding too long a duplex at replication at one time seemed to m a k e the linker hypothesis more l i k e l y .1 4 4
T h e situation was changed, however, when it was found t h a t shearing forces, such as are applied to D N A in solution by mere pipetting, are capa
ble of breaking long molecules near their c e n t e r .1 4 5 - 1 4 8 I t <was found t h a t carefully extracted D N A from phage T 2 had a molecular weight as high as 1.3 Χ 108, representing the total D N A of the phage c h r o m o s o m e .1 4 9 - 1 5 1
T h e following conclusions were drawn from the results :
1. Even D N A from the chromosome of a bacterium or of an animal cell might also be a single enormous molecule, much too long to be possibly studied intact in solution.
2. There is no longer any need to assume the existence of linkers, since the unwinding difficulties are not so serious after all.
A word must be said of the techniques employed for measuring the molecular weight of D N A . The usual ones (involving light-scattering or sedimentation measurements) cease being reliable for weights above
16 Χ Ι Ο6 1 5 2'1 5 3 ; this is not true, however, of the autoradiographic m e t h o d .1 5 4 Electron microscopy is another powerful tool, permitting an estimation of length distribution among the molecules.1 5 5 When these more appropriate techniques were applied to bacterial D N A , liberated from the cells by specially mild procedures, a minimal molecular weight of 1 Χ 109 was o b t a i n e d1 5 6 and the electron micrographs were consistent with D N A being present as one continuous t h r e a d .1 5 7 If one also remembers t h a t the best-known bacterial genome, t h a t of E. coli, behaves genetically as one linear s t r u c t u r e ,1 5 8 the presumption becomes very strong t h a t the 50 molecular species one is dealing with in transformation are an artifact.
T h e fragility of the native chromosomal D N A in solution is such, however, t h a t artifact cannot be avoided.
C. B A S E COMPOSITION AND I T S DISTRIBUTION AMONG INDIVIDUAL D N A MOLECULES
As a result of the amounts of adenine and thymine and of guanine and cytosine (A, T, G, and C, respectively) being e q u i m o l a r ,2 1'1 3 7 one base content is enough to characterize the mean composition of D N A . (The latter is often expressed as per cent of G + C, i.e., 2 G % ) . I n bacteria the G-C content is found to vary widely (from 25 to 75%) with the species and m a y reflect phylogenetic r e l a t i o n s1 5 9 - 1 6 1; see Chapter 9). Several physical constants, such as the d e n s i t y1 6 2 - 1 6 4 and the melting temperature,
1 0 6 P I E R R E SCHAEFFER
Tm ,1 6 5 were shown to vary linearly with the guanine content; these obser
vations made it possible to estimate the composition of a D N A sample from its Tm v a l u e ,1 0 0 or from a density d e t e r m i n a t i o n1 6 7 requiring merely an equilibrium sedimentation of the solution in a CsCl density g r a d i e n t .1 6 8 Additional information can be obtained with these physical methods: the sharpness of the melting reflects the compositional heterogeneity of the D N A preparation and the width of the D N A band in the gradient, its polydispersity. Although bacterial D N A ' s show a relatively small dis
persion of the G-C content among their molecules, it has been possible to establish by centrifugation t h a t D N A particles of different densities carry distinct m a r k e r s .1 6 9'1 7 0 While the physical heterogeneity of D N A was thereby clearly demonstrated, no clean separation of specific molecules could be obtained. Similar conclusions had already been reached in studies in which fractionation of transforming D N A had been attempted by chromatographic t e c h n i q u e s .1 7 1'1 7 2 Better results were claimed by Bendich et a L ,1 7 3 , 1 7 4 obtained by a procedure t h a t has not been adopted by other workers. Lerman's columns (kieselguhr impregnated with methyl
ated albumin), as employed by Hershey et al.,145'175 and by Sueoka and C h e n g ,1 7 6 do fractionate D N A according to size, composition, and second
ary structure, however. The D N A of the defective, transducing phage Adg is a case of a homogenous transforming D N A ,7 3 but this is only so because no fractionation is required in this case.
A similarity in mean composition of the D N A ' s of the two parental strains has generally been recognized to be a minimal requirement for t r a n s f o r m a t i o n6 3'1 1 8 , 1 3 9 and more generally for genetic recombination.1 7 7 The same is true for the formation of hybrid molecules by renatura- t i o n ,1 7 8 - 1 8 0 but no correlation was found to exist between the composition of a D N A and its activity as an inhibitor of transformation.1 3 9
D . D N A REPLICATION
1. T H E FORMAL A S P E C T
The complementarity of the strands in the Watson-Crick structure led these authors to propose t h a t each strand might function as a template for the synthesis of the other (see Chapter 7 ) . The strand separation im
plied in such a model seemed at first to run into topological and energetic difficulties, which m a y be more apparent t h a n real, however.1 4 8 Also, as pointed out by Delbriick and Stent, the proposed replication mechanism leads to the prediction t h a t the single strands of the parental D N A duplex will become separated from each other at the first replication, while con
serving their atomic identity in this and the subsequent replication; in others words, replication is predicted to be semiconservative with respect to the distribution of the atoms of the parental to the daughter molecules.1 8 1
T h e experimental verification of this prediction, achieved by Meselson and S t a h l1 8 2 and by Levinthal and T h o m a s ,1 8 3 strengthened the hypothesis considerably ; complete demonstration, however, would require the identi
fication of the conserved subunits with single nucleotide chains, and this has not been d o n e .1 8 2 These subunits (shown by Rolfe to be associated lat
e r a l l y ,1 8 4 as assumed in the model), were claimed by Cavalieri et al. to consist of two chains rather t h a n one, the parental molecule itself being f o u r - s t r a n d e d .1 8 5 - 1 8 7 This claim has far-reaching implications, since it leads to the rejection of the concept t h a t base-pairing plays a role in dupli
cation, but it also has physical implications which seem to be already d i s p r o v e d .1 8 8 , 1 8 9 T h e following points in Cavalieri's work seem more likely to be generally accepted: (!) D N A is in different physical states, depend
ing on whether the cells from which it was extracted were actively dividing or not; (2) heat treatment of D N A in concentrated CsCl solution differ
entiates the two states; (3) D N A goes through the nondividing state during a small fraction of the division cycle, as can be shown with synchro
nized c u l t u r e s1 8 5; (see also E p h r u s s i - T a y l o r1 9 0) .
2. T H E BIOCHEMICAL A S P E C T
As is well known, in vitro synthesis of D N A has been obtained in R o m berg's l a b o r a t o r y5 2 and shown to correspond to the following reaction :
d C T P J + Mg
in which dXTP is a deoxynucleoside triphosphate.
I n this enzymic reaction D N A is required as a p r i m e r ,5 3'1 9 1 both strands of which are copied. No increase in activity has so far been obtained, when a transforming D N A is supplied as primer.* Single D N A chains are en
dowed with priming activity and there are reasons to believe t h a t a double- stranded D N A will be active only if it has first somehow been dena
t u r e d .1 9 1 , 1 9 2 Synthesis does not stop, when an amount of D N A equal to the primer has been made.
I n conclusion, any participation in D N A replication of R N A , or of a specific protein other t h a n an enzyme, seems excluded by the biochemical studies; the biosynthetic reaction described is compatible with the semi- conservative model of Watson and Crick.
3. D N A SYNTHESIS AND THE D I V I S I O N CYCLE
While in cells from higher organisms D N A synthesis is restricted to the early prophase, it has been found in bacteria to extend over most, and possibly all, of the division cycle. This has been shown in a variety of
* N o t e a d d e d o n proof : See Chapter 7, h o w e v e r . DNA polymerase
+ primer DNA + Mg++
:t D n a + i n o r g a n i c p y r o p h o s p h a t e
108 P I E R R E SCHAEFFER
w a y s .1 9 3 - 1 9 5 Once started in a cell, D N A replication, according to Maal0e, is independent of protein (and R N A ) synthesis; but some protein synthesis must have taken place, before the next replication cycle can start again.
The proposed interpretation is, t h a t D N A replication starts at one point and proceeds from there on, all along the chromosome.1 9 6
E . HETEROCATALYTIC ACTIVITY OF D N A
The transfer and recombination of genetic information and the func
tioning of this information are two distinct fields of study. Transformation proper can be considered terminated with genetic integration, the only borderline problem being whether integration of a newly introduced piece of D N A is a prerequisite to its function. For the mechanism and specificity of protein synthesis, the reader is referred to Chapter 8.
F . ALTERATIONS OF THE D N A MOLECULE
I t has long been realized t h a t transformation offered the unique ad
vantage of the possibility of altering the structure of the D N A molecule by various agents, and of then relating the nature and extent of the lesion with biological activity. Nevertheless, such experiments involve consider
able difficulties. I n the first place, the agents applied m a y cause more than one type of lesion. T h e precise determination of the nature and extent of the various lesions and the complexity of the biological test, transforma
tion, in which different steps m a y be sensitive to different types of struc
tural alterations, pose additional problems.
1. H E A T TREATMENT
To quote an early work, the transforming activity of pneumococcus ex
tracts is unaffected by heating 30 minutes at 6 0 ° C , reduced abruptly a t 8 0 ° C , and some residual activity remains even after 10 minutes a t 90°C.
This unexpectedly high thermoresistance had first been reported by Allo- way in 1933,1 5 but it is only recently t h a t the facts could be interpreted in
terms of molecular changes. Today, largely as a result of the outstanding contributions of Meselson and S t a h l ,1 8 2 D o t y et al. (cf. réf. 152), Lerman and T o l m a c h ,1 9 7 Roger and H o t c h k i s s ,1 9 8 etc., two distinct types of heat inactivation have been recognized. Their description requires t h a t the notion of denaturation be first introduced.
a. Denaturation. When D N A solutions are heated for a short time at tem
peratures close to boiling and cooled rapidly, all their properties have changed. The transforming activity is reduced to a few per cent (residual a c t i v i t y1 9 8) , the viscosity d r o p s ,1 7 , 1 9 9 the relative absorbance at 260 m/x in
creases (hyperchromic e f f e c t ) ,2 0 0'2 0 1 the buoyant density increases,1 8 2 , 2 0 2
the reactivity to certain chemical and physical agents (formaldehyde, UV)