SOME NEW DATA CONCERNING THE MUTAGENIC ACTION OF DNA

// is shown that spermatozoa of Drosophila melanogaster can transfer molecules of exo­ genous DNA (or their fragments) from a solution of this DNA injected into the haemocel of an adult male into the ooplasm of the egg. As a result, mutations induced in the descendants arise both in the paternal and the maternal chromosomes obtained by them. The mutagenic effect of exogenous DNA has a prolonged character inducing mutations in the descendants during many cell generations after DNA-treatment of their father. The mechanism of such a prolongation of the mutagenic action of DNA is discussed.

// is shown that spermatozoa of Drosophila melanogaster can transfer molecules of exo genous DNA (or their fragments) from a solution of this DNA injected into the haemocel of an adult male into the ooplasm of the egg.As a result, mutations induced in the descendants arise both in the paternal and the maternal chromosomes obtained by them.The mutagenic effect of exogenous DNA has a prolonged character inducing mutations in the descendants during many cell generations after DNA-treatment of their father.The mechanism of such a prolongation of the mutagenic action of DNA is discussed.
In our previous experiments [1,2] on the induction of recessive lethal mutations in the 2nd chromosome of Drosophila melanogaster by injec tions of a solution of exogenous DNA into the haemocel of adult males we found that the frequency of induced lethal in the progeny which deve loped from eggs laid by the female fertilized by a DNA-treated male du ring the first three days after copulation was in most cases only slightly different from the frequency of such mutations in the progeny which de veloped from eggs laid later.This surprised us as it meant that DNA can induce mutations not only in the dividing premeiotic germ cells of the males but also in their completely formed ripe spermatozoa.This was later confirmed by the results of an experiment in which DNA-treated males were immediately after their first copulation separated from the females with which they have copulated, so that we could be certain that the eggs laid by the females were fertilized by spermatozoa which were in contact with exogenous DNA already being ripe.The frequency of lethals induced by DNA was here again significantly higher than in the untreated control.
In seems utterly unbelievable that very large DNA molecules present in the solution injected into the haemocel of the male can penetrate into the tightly compressed nucleus forming the head of the spermatozoan.Much more probable is that DNA molecules are adsorbed on the surface of the spermatozoan and thus are purely mechanically transported and introduced into the ooplasm of the fertilized egg where they reach both.the male and female pronuclei and eventually the paternal and maternal chromosomes in them.
If this supposition is correct then exogenous DNA injected into the male should induce mutations in chromosomes received by the descen dants both form their treated father and their untreated mother.In fa vour of this supposition speaks the experiment described in 1971 by Brackett et al. [3]; it showed that mammalian spermatozoa can introduce into the egg DNA molecules adsorbed by them from the surrounding fluid.Recently this has been confirmed by Lavitrano et al. [4] and by Siracuso et al. [5]; their experiments showed, for example, that murine spermato zoa, incubated in an isotopic buffer containing an exogenous DNA (the plasmid pSV2 CAT) are introduced into eggs fertilized by them.Embry os thus obtained were implanted into pseudo-pregnant females and among 250 descendants of these females about a third contained in their genomic DNA nucleotide sequences of the above-mentioned plasmid.And when transgenic females were crossed with CD1 males the gene CAT was ex pressed in the bones and tails of their descendants.In all, these authors had proved that such a transport of exogenous DNA into the ooplasm can be achieved by spermatozoa of mice, bulls and man; and it is quite probable ihat this can place in insects.
To check this hypothesis we carried out an experiment using as mar kers the following mutant genes located in the 2nd chromosome of D. melanogaster -Curly (Cy), Bristle (Bl), Lobe 2 (L 2 ), black (b) and cin nabar (en).Calf thymus DNA was dissolved in saline and injected (ca.Ю.07-0.09micrograms of DNA in 0.25 ml of fluid per male) into the haemocel of heterozygous males having one of its 2nd chromosomes mar ked with Cy and Bl and the other with en (Cy Bl/cn).These males were crossed with virgin heterozygous females having one of its 2nd chromo somes marked with Cy and the other with b(Cy/b).The Fi from these crosses consisted of flies including two Curly classes, Cy/cn and Cy/b which can be easily distinguished one from another.Each of the Fi Cy Bl/b males was individually crossed with a virgin Cy/L 2 female and their F 2 descendants were inbred, this allowing to detect in the F 3 recessive lethal mutations which had arisen in the 2nd chromosome of their untre ated grandmother (in this case no black flies will be present in the F 3 ).Likewise, each of the Fi Cy/cn males was individually crossed with a vir gin Cy/L 2 female and their F 2 descendants were inbred, this allowing to jdetect in the F 3 recessive lethal mutations which had arise in the 2nd chromosome of their DNA-treated grandfather (in this case no cinnabar flies will be present in the F 3 ).
The results of this experiment is presented in Table 1.As shown in this table, the frequency of recessive lethal mutations in the paternal and the maternal chromosome is about the same, exceeding about ten times the frequency of such mutations which spontaneously arose in the untre ated control.This allows us to be rather certain that the induced mutati ons arose not in ripe spermatozoa but in the chromosomes of both pro nuclei (paternal and maternal) contained in the fertilized egg.(A similar experiment was performed on lethals induced in the 2nd chromosome by the synthetic polyribonucleotide poly (A, U).This experiment also showen that the mutagen can be carried by spermatozoa to the female pronucleus.The number of lethals induced in the paternal (5.73 %f) and the maternal (4.50 %) chromosome of Fi flies was nearly equal.)However, as this experiment was carried out on a relatively modest scale this conclusion is only preliminary and needs a repetition on a lar ger scale.
In one of the experiments of Fahmy and Fahmy [6].. in which exoge nous DNA was injected into adult Drosophila males, a statistically sig nificant increase was observed among Fi females of the mutability of the garnet locus in the X chromosome received from their mother and not from the DNA-treated father.These authors assume that here the exo genous DNA injected into the father was passively transmitted by the spermatozoa to his daughters.We are inclined to regard this case as speaking for the correctness of our conclusion based on the data presented in Table 1.Already in our first experiments [7] on the induction of mutations in D. melanogaster by exogenous DNA it was found that the mutagenic action of DNA is often not immediate but considerably delayed so that many mutations in the Fi of DNA-treated males appear as mosaic indi viduals.As was shown by these experiments and numerous later ones, some gene mutations induced by exogenous DNA added to the food of D. melanogaster larvae or injected into the haemocel of adult males ap pear in the Fi as buches of mutants (this showing that the arose in premeiotic germ cells of the male) or as whole (non-mosaic) individuals but many of them, usually more than 50 per cent, appear as mosaic in which only 1/2, 1/4 or a lesser part of their body consists of mutant tis sues.For example, in the Fi of some such experiments among 13 247 flies 27 visible gene mutations were found twenty of which appeared as mosa ics (chiefly 1/2 or 1/4 mosaics).This group included flies manifesting phenotypical mosaicism (e. g. with one normal and one miniature wings) which had mosaic gonads; and also it included some phenotypically whole mutant (non-mosaic flies which had mosaic gonads [8]).On Table 2 are shown the results of crossing such Fi mosaic males with virgin attached-X females.Other mosaic males in this and other similar experiments gave analogous results.To the 20 mosaic flies in this experimental series a number of phenotypically mosaic flies probably should be added which gave no offspring or gave only wild-type descendants and therefore were not registered as mutants.

Recessive lethal mutations induced in the 2nd chromosome of Drosophila melanogaster in experiments on transportation of DNA molecules by spermatozoa into the egg
A preponderance of mosaic mutants over whole ones was characteris tic not only of sex-linked visible mutations but also of autosomal ones •and was observed in our experiments not only in the Fi but in the F 2 and F 3 as well.
Many Fi mosaics in which «the mutant part was ca 1/4 of the body |or less must have arisen as a result of mutation which took place two or more cell generations after the direct treatment of DNA had stopped.But the mutagenic effect of DNA may be manifested even later.We have shown in our experiments that the mutagenic effect of DNA is evident not only in the Fi but also in the F 2 and the F 3 though in the latter it is somewhat weaker; so it is still present several dozens of cell generations after the cessation of the treatment.
In some cases the same gene mutation arose in different generations of descendants originating from a single treated male.Thus, in one of our experiments a mosaic miniature male was found in the Fi; this male was crossed with an attached-X female and some of their sons were mi niature and other had wild-type wings, this being a result of mosaicism of the father's gonads.One of the wild-type sons had deformed eyes and in order to analyse this deformity the male was crossed with an atta ched-X female.The deformed eye proved to be non-hereditary but among the offspring of this cross a single mosaic miniature male again appeared from which a pure miniature stock was later established.A similar pic ture was observed in the progeny of a mosaic rudimentary male and ru dimentary mosaics and whole rudimentary males continued to appear untill the F 6 .A pedigree of this family is given in Fig.
In another of our experiments an Fi male was a fused mutant.In the offspring of its wild-type male brother again a fused mutant was fo- und; it was a mosaic both in respect of wings and gonads.Such cases show that treatment with exogenous DNA leads to the appearance of a transmittable unstability of certain genes.
In one case the mutant nature of an F { fly could be ascertained only because it was a mosaic.This fly, a male, carried a dominant autosomal mutations which we named Beaded-crossveinless (its phenotype remin ded both the well known Beaded and the crossveinless mutations of D. melanogaster).When crossed with an attached-X female it gave a pro geny consisting of 67 flies among which 9 females and 11 males were Part of a pedigree in which rudimentary mutations appeared among the descendants of a mutant male in which the mutation was induced by exogenous DNA Beaded-crossveinless.Both saxes of these mutants had underdeveloped gonads and were completely steril.Evidently, their mutant father has been a mosaic the fertility of which was due to the presence in his body of normal non-mutant tissues.
We did not study mosaicism among recessive lethal mutations indu ced by DNA but Mathew [9] and Khan and Alderson [10] used a gene tic technique which allowed to detect in such experiments not only com plete (non-mosaic) lethals but mosaic ones as well.The proportion of •mosaic was here even higher than in our experiments on the induction of visible mutations.In many treated lines Fi mosaics again produced mosaics in the F 2 and so on, up to F 5 (in the experiments of Khan and Alderson) and even to F 9 (in experiments of Mathew).The extension of the mutagenic effect of DNA to such late generations strongly supports the supposition made above about a transmittable destabilization by exo genous DNA of certain genes.
Fahmy and Fahmy [6,11] found mosaics among visible recessive mutations and Minutes induced by injection into adult D. melanogaster males of DNA isolated from larvae of the same species and from rat liver.
The data obtained in our previous work the induction of visible and lethal mutations in D. melanogaster by exogenous DNAs showed that these mutations in many important aspects closely resemble mutations in duced by spontaneous insertions into the chromosomes of the recipient of mobile genetic element of several kinds [12][13][14][15] and many other.It is most unlikely that this parallelism of the peculiarities of the mutagenic effects of spontaneous insertions of mobile genetic elements and of injec tions of exogenous DNA is accidental.It seems much more probable that fragments of molecules of exogenous DNA act like transpositions of mo bile genetic elements becoming inserted into chromosomes and selecti vely altering or destabilizing certain genes.If this hypothesis is correct it explains the prolonged mutagenic action of exogenous DNA.Fragments of this DNA for a time remain in a free state, like episomes, in the cells of the recipient and are thus transmitted from one cell generation to another; or they can be inserted into the DNA of chromosomes this cau sing gene mutations or inducing minor chromosome rearrangement this behaviour resembling that of some mobile genetic elements, e. g. copia (Flavell and Ish-Horowicz [16], Shiba and Saigo [17], Yamafumi et al. [18] and other authors).
The reversions to wild-type observed by us of some unstable muta tions induced in Drosophila by exogenous DNA may be caused by an ex cision of the inserted fragment of foreign DNA or by a change of its ori entation within the chromosome.