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gratitude to Drs. M. M. Rapport and L. Graf for their generous assistance and for making availa-ble many of the substances used in this investigation.

* The work reported in this communication was for the most part performed in the laboratoriesof The Rockefeller University, New York.

1 Tal, C., T. Dishon, and J. Gross, Brit. J. Cancer, 18, 111 (1964).2 Rapport, M. M., L. Graf, V. P. Skipski, and N. F. Alonzo, Nature, 181, 1803 (1958); Cancer,

12, 438 (1959).3 Rapport, M. M., L. Graf, and J. Yariv, Arch. Biochem. Biophys., 92, 438 (1961).4 Shapiro, D., and E. S. Rachaman, Nature, 201, 878 (1964).5 Rapport, M. M., L. Graf, and H. Schneider, Arch. Biochem. Biophys., 105, 431 (1964).6 Graf, L., and M. M. Rapport, Cancer Res., 20, 546 (1960).

CHANGES IN CHROMOSOMES INDUCED BY MICROORGANISMINFECTION*

BY M. DIAZt AND C. PAVANtINSTITUTO DE INVESTIGATION DE CIENCIAS BIOLOGICAS, MONTEVIDEO, URUGUAY,

DEPARTAMENTO DE BIOLOGIA GERAL, UNIVERSIDADE DE SAO PAULO,AND BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE

Communicated by Alexander Hollaender, September 21, 1965

Changes in cell physiology are the primary consequences of any infection. Occa-sionally, however, infection causes changes in the genetic material of the cells. Thefinding in tissues of Rhynchosciara angelae (Diptera, Sciaridae) of two microbialinfections that produce general changes in the nucleus as well as changes in specificloci of the chromosomes is the subject of this communication. R. angelae haspolytene chromosomes (A, B, C, and X) in several tissues of the larva as well as theadult (Figs. 1-9).'1-

Infection by a Protozoan.-In one of our experiments with R. angelae we found alarva in which four cells of the salivary gland were infected by a protozoan. Themicroorganism was found in large numbers in each of the infected cells. It existedin at least two different forms. One form was spherical, about 3 ,u in diameter, had acentral nucleus about 1 1A in diameter, and was normally found in groups of eightattached together. The other form was ellipsoid, with axes of about 3 and 7 IA; thisform was found free inside the cell without any aggregation. In this phase thenuclei did not acquire the cresyl-violet stain, but did incorporate H3-thymidine thatwas injected into the larva 2 hr before it was killed.The infected cells showed typical signs of alteration, the most evident being an

increase both in total cell size and in size of the chromosomes (Figs. 1-5). Thenormal cells, even those located at the edge of the infected ones, showed no sign ofalteration. Besides the increase in chromosome size, some specific changes werealso produced in the heterochromatin region. The heterochromatin in the polytenechromosomes of R. angelae is normally distributed near the centromeric regions ofthe four chromosomes3 (Fig. 2). The nucleolar organizer region is located near theheterochromatic portion of the X chromosome. In the infected cells, the hetero-chromatic regions of the chromosomes were the most affected, and in one of the cells(Fig. 1) each of the heterochromatic blocks was transformed into a structure which

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1322 GENETICS: DIAZ AND PAVAN PROC. N. A. S.

,+>S_ 1 _ i~~~~~~~iA p0 W *F Seiko trio *s~~~~~~~~~~~F..........

.. .......

PAX ~ ~ ~~~~~~J.WDo .44 trs,*E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... #oi......

io,

,,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~e

8^~~~~~~~~~~4; i-b,@Zl

x

B,

FIGS. 1 and 2.-Photomicrographs of the polytene chromosome set (A, B, C, and X) of cells ofthe salivary gland of Rhynchosciara angelae, one infected with a protozoan (Fig. 1) and one normal(Fig. 2). The arrows indicate the heterochromatic regions, located in a median region of chromo-some A and at the base of the other chromosomes. In the infected cells, the heterochromaticregions (with the exception of the one in chromosome A of this particular cell) are transformedinto structures which are a combination of a large puff and a nucleolus. The nucleolar organizerregion is located in the base of chromosome X, but in cells of the salivary gland of old larvae,as can be seen in Fig. 2, no evident nucleolus is observed. The great number of the dark spotsfound in the background of Fig. 1 corresponds to the protozoan specimens. The scale correspondsto 200 u for both figures.

had characteristics both of a large puff and of a nucleolus. The heterochromatinwas dispersed, forming a net of DNA fibrils having among them an amorphous massof material that resembled very much the RNA and protein of the nucleolus. Therest of the chromosomes in the infected cells showed bands less condensed than innormal chromosomes. The appearance of these bands and interbands indicatedthat there was a high activity in these regions, but this transformation was not sostriking as those that took place in the heterochromatic portion. The incorporationof H3-thymidine into the chromosomes of infected cells showed that they were stillactive, and not degenerating.

In Diptera there are at least two other known cases of protozoan infections thatpromote changes in the karyotype of the infected cells. One of them is an infectionin the cells of the salivary gland of Chironomus anthracinus by a microsporidian:infected cells show hypertrophy both in total cell size and in size of the chromosomes.These hypertrophied chromosomes are very similar to normal ones, however, ingeneral shape and banding pattern.4 The other case reported is an infection in thecells of the salivary gland of Simulium maculata (Diptera) by Thelohaniafibrata, alsoa microsporidian, which promotes hypertrophy of the cell and of its nuclei.5

Virus Infection.-In one group of R. angelae we observed several individuals whichwere delayed in development, were smaller than the rest of the colony, and hadblocks of opaque material located in the wall of a pair of long glands (gastric caecae)

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VOL. 154, 1965 GENETICS: DIAZ AND PAVAN 1323

50.P~...AFIGS. 3, 4, and 5. Photomicrographs of chromosome C of cells of R. angela, one normal and

one infected with a protozoan. Figs. 3 and 4 are from the same chromosome; Fig. 3 is for com-parison of corresponding regions, and Fig. 4 is for comparison of size with the chromosome fromthe infected cell (Fig. 5). The nucleolus-like structure found in the heterochromatic region of thechromosome from the infected cell was never found in chromosomes of normal cells. The upperscale corresponds to 50 js and is valid for Fig. 3; the lower corresponds to 200 is and is valid forFigs. 4 and 5.

that are attached to the crop and run parallel to the median intestine. In prepara-tions from acetic orcein squashes we observed that the cells of the gland were hyper-trophied, forming small tumors. The opacity of the tumors was caused by greatnumbers of polyhedral crystals 1-14 ,u in diameter. The largest crystals showedslightly rhombohedral contours. We could not determine the exact localization ofthese inclusions in the cells because we made only squash preparations. In Feulgen-stained preparations, the inclusions showed a homogenous light pink coloration. Inthis infection also, increase in the size of cells was accompanied by an increase in sizeof the chromosomes (Figs. 6 and 7). In the larvae which we examined it was possi-ble to find cells in many stages of infection, as well as normal cells among them. Inaddition to their abnormally large volume, the chromosomes of infected cells showedspecific changes in banding pattern. Some chromosomes exhibited light regionswhich resembled the one found normally in section 9 of chromosome A.2 We don't

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1324 GENETICS: DIAZ AND PAVAN PROC. N. A. S.

know the meaning of these light regions, but we know that they do not appear inchromosomes of the normal cells of the salivary gland, of the Malpighian tubules, orof the anterior intestine. In the largest infected cell, which also had the largestchromosomes, typical signs of degeneration of the genetic material were observed(Fig. 8). The most striking was the fragmentation of the chromosomes, but alsobridges between two fragments, as well as some vacuolization, were observed incertain parts of the chromosomes.

In some cases the chromosomes were surrounded by a homogenous mass ofFeulgen-positive material. Autoradiographs made after H3-thymidine injectionshowed that the polytene chromosomes of infected cells incorporated the radioactiveprecursor very readily, but a high rate of incorporation was found also in other partsof the cells outside of the chromosome. A microspectrophotometric measurement ofa Feulgen preparation' showed that in the moderately hypertrophied cells, in whichno inclusions were present yet, the mean value of the DNA content of the polytenechromosomes was about 7.6 times higher than normal.The appearance of these inclusions fits the description of polyhedrosis, a viral

disease of insects. The Feulgen-positive reaction in the inclusions as well as theincorporation of H3-thymidine on the surface of the inclusions and in other regionsoutside the chromosomes are good indications that this hypertrophy of the cells iscaused by a DNA virus.

In the known cases of nuclear polyhedrosis the production of inclusions is alwaysaccompanied by nuclear hypertrophy,7 but in no case has chromosomal hypertrophyor endomitosis been observed. There is some indication, however, that somethingsimilar may happen in cells of Bombyx mori after infection with polyhedral virus.8-"1

Discussion.-Biological agents that cause specific changes in the karyotype of allaffected cells of an organism, such as we have found in R. angelae, can be used tostudy the problem of normal and abnormal differentiation. Recent results obtainedin studies on the interaction of viral and bacterial genome and the relation of someviruses and the karyotype of the host in higher organisms12' 13 make the findings inR. angelae of special interest. Both infections that we found produce specificchanges in the karyotype of the affected cells, and in both cases the changes lead thecells to a different development. These changes are perhaps a particular case of amore general process that may occur in intracellular infections of several organismscaused by different infective agents. There are several reported cases of changes inthe morphology and the physiology of the nucleus after the development of micro-organisms inside the mammalian cell. Cases have been described in which viral in-fections cause chromosomal aberrations.12-22 In some cases the induced changesappear to affect preferentially some chromosomes of the genome. 17, 18 The results ofHare and McFeely,23 who have found hypertrophied chromosomes in a few cells of anormal dog, indicate that the changes of the type observed in R. angelae may welloccur in cells of mammals. No infective agent has been implicated in this instance,however. Changes in the internal environment of the cell caused by agents otherthan infection may determine changes in the genetic constitution of the cell. Ger-stel and Burns24 found one or a few chromosomes of unusual length among thenormal chromosomes of some cells of hybrids between two species of Nicotiana.Changes in the banding pattern of a particular region, the polytene chromosomes ofDrosophila pseudoobscura, are induced by a recessive gene (salivary).25

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VOL. 54, 1965 GENETICS: DIAZ AND PAVAN 1325

..¶~~~~~...*~~~~~~~~~~~~~~~~~~.X....

A A

*.

44

_s ~~~~~~~~~~~~~~~~*

A. ,

.,:.. A: f........;_-.....^

FIGs. 6 and 7.-Photornicrographs of polytene chromosomes of infected (with DNA virus)and normal cells of the gastric caeca gland of R. angelae. The larger chromosomes are frominfected cells and the smaller from normal cells of the same gland. As the preparation is a squashof the gland in acetic orcein, chromosomes from different nuclei often come together. The darkspots of different sizes correspond to polyhedral crystals which are produced by the cell afterbeing infected by the virus.

FIG. 8.-Photomicrograph of polytene chromosomes of heavily infected cells of gastri caecagland of R. angelae. The black spot in the background corresponds to the polyhedral crystalswhich were very abundant in this cell. The chromosomes show typical signs of degradation,breaks, gaps, and overcondensation of the chromatin.

FIG. 9.-Photomicrograph of an entire set of polytene chromosomes of a normal cell of thegastric caeca gland of R. angelae. The scale corresponds to S0 p and is valid for Figs. 6-9.

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1326 GENETICS: DIAZ AND PAVAN PROC. N. A. S.

The chromosomal changes discussed above involve somatic cells, and are of greatimportance for special studies in pathology and in abnormal development. Similarchanges in the cells of the germline, however, could be of great importance for thephylogeny of the species. Very interesting in relation to this are the results ofLevitan and associates26- in experiments with Drosophila robusta, in which theyfound a maternal factor that produces a high frequency of chromosome aberrations.The new chromosomal rearrangements are then transmitted to the next generation.We have enough evidence showing that the behavior as well as the constitution of

the cell karyotype can be changed under the influence of the environment in which itoperates. Infective microorganisms change the internal environment of the cellsand are very efficient inducers of karyotype changes in the host cell, and in somecases the changes are specific to the agent which induces them. The study of infec-tions in cells having polytene chromosomes enables us to know in a more precise waywhich are the main reactions of the chromosome loci to an infective agent. Know-ing this, we will have a better base to interpret the reactions of a diploid cell toinfective agents, as well as a better understanding of the process of differentiation.

The authors are grateful to the Directors of the Rockefeller Foundation (which made possibleMr. Diaz' trip to Sdo Paulo and maintenance there for four months), the National Institutes ofHealth, and the Fundagdo de Amparo a Pesquisa do Estado de Sdo Paulo. They also wish tothank Drs. A. Brito da Cunha, R. F. Kimball, and D. L. Lindsley, Jr., for criticism and sugges-tions, and Mr. D. Amundson for editorial assistance.

* This research was jointly sponsored by the U.S. Atomic Energy Commission under contractwith the Union Carbide Corporation, by the Rockefeller Foundation, by the National Institutes ofHealth (contract G.M. 11493-01), and by the Fundagdo de Amparo a Pesquisa do Estado de SdoPaulo (FAPESP).

t Instituto de Investigation de Ciencias Biologicas, Montevideo, Uruguay.t Departamento de Biologia Geral, Universidade de Sdo Paulo; and Biology Division, Oak

Ridge National Laboratory, Oak Ridge, Tennessee.1 Dreyfus, A., E. Nonato, M. E. Breuer, and C. Pavan, Rev. Brasil. Biol., 11, 439 (1951).2 Pavan, C., and M. E. Breuer, J. Heredity, 43, 150 (1952).3 Breuer, M. E., and C. Pavan, Chromosoma, 7, 371 (1955).4 Keyl, H. G., Naturwiss., 47, 212 (1960).5 Debaisieux, P., Compt. Rend. Soc. Biol., 82, 867 (1919).6 Diaz, M., and M. E. Drets, unpublished observations.7 Xeros, N., Biochim. Biophys. Acta, 55, 176 (1962).8 Gratia, A., J. Brachet, and R. Jeener, Bull. Acad. Roy. Med. Belg., 10, 72 (1945).9 Semenova, Z. A., Ph.D. thesis, Moscow (1951).10 Yamafugi, K., M. Shimamura, and F. Yoshihara, Enzymology, 16, 337 (1962).11 Morris, 0. N., J. Insect Pathol., 4, 454 (1962).12 Koprowski, H., J. A. Pont6n, F. Jensen, R. G. Ravdin, P. Moorhead, and E. Saksela, J. Cell.

Comp. Physiol., 59, 281 (1962).13 Cooper, H. L., and P. H. Black, J. Natl. Cancer Inst., 30, 1015 (1963).14 Yerganian, G., H. M. Shein, and J. E. Enders, Cytogenetics, 1, 316 (1962).16 Nichols, W. W., A. Levan, B. Hall, and G. Ostergen, Hereditas, 48, 367 (1962).16 Aula, P., Hereditas, 49, 451 (1963).17 Moorhead, P. S., and E. Saksela, J. Cell. Comp. Physiol., 62, 57 (1963).18 Wald, N., A. C. Upton, U. K. Jenkins, and W. H. Borges, Science, 143, 810 (1964).19 Hampar, B., and S. A. Ellison, Nature (London), 192, 145 (1961).20 Stich, H. F., G. L. van Hoosier, and J. J. Trentin, Exptl. Cell Res., 34, 400 (1964).21 Makino, S., K. Yamada, and T. Kajii, Chromosoma, 16, 372 (1965).22 Dulbecco, R., and M. Vogt, these PROCEEDINGS, 46, 1617 (1960).23 Hare, W. C. D., and R. A. McFeely, Mammalian Chromosome News Letters, 13, 3 (1964).

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24Gerstel, D. U., and J. A. Burns, unpublished observations.25 Levine, L., and L. van Valen, Science, 137, 993 (1962).26 Levitan, M., these PROCEEDINGS, 48, 930 (1962).27 Levitan, M., Nature (London), 200, 437 (1963).28 Levitan, M., and R. Schiller, Genetics, 48, 1231 (1963).

LOW FREQUENCY OF CO-INTEGRATION OF TWO LINKEDSTREPTOMYCIN-RESISTANCE MARKERS IN

PNEUMOCOCCUS*

BY MINNA B. ROTHEIM AND ARNOLD W. RAVIN

DEPARTMENT OF BIOLOGY, UNIVERSITY OF ROCHESTER

Communicated by M. Demerec, September 16, 1965

It has been shown1' 2 through mapping with nonidentical, overlapping multisitemutations that all streptomycin-resistance (str-r) mutations are located within thesame gene locus of the pneumococcal genome. At the same time, however, it wasobserved2 that when a single donor DNA preparation containing the mutationsstr-r42 and str-r2 was used to transform streptomycin-sensitive recipients, twomolecules of DNA were necessary to produce a doubly marked (str-r42-r2) trans-formant; that is to say, when sensitive cells were exposed to different concentra-tions of transforming DNA prepared from a doubly marked str-r42-r2 strain, thefrequency of double transformants increased as the square of the increase in DNAconcentration. The apparent independence of the str-r42 and -r2 markers intransformation meant that a break occurred in the region between the two markerssometime during the extraction of and subsequent transformation by the doublymarked DNA. Two possible explanations could be offered: either the process ofpurification and extraction of DNA resulted in breakage between str-r42 and -r2 or,alternatively, the probability of co-integration of the two markers from the samemolecule was highly unlikely.The data to be presented here show that the latter is correct. If different batches

of sensitive recipient cells are treated with varying concentrations of the samedoubly marked str-r42-r2 DNA, keeping all other experimental conditions constant,direct proportionality between the number of doubly transformed cells and DNAconcentration may or may not be observed depending on the physiological state ofthe competent cells used.

Materials and Methods.-Strains: All media, methods of transformation, andmost of the strains have been previously described.2-4 The genetic relationshipbetween all the str-r mutations is shown in Figure 1. The procedure by which thismap is made has been described.1' 2 In addition to these str-r mutations, an eryth-romycin-resistance marker, ery-r2,6 was used. The sulfanilamide-resistancemarker Fd (or sulfa-rd) was obtained via a transforming DNA preparation kindlysupplied by Dr. R. Hotchkiss.6

Preparation and storage of competent cells: A slight modification in the pro-cedure4 for preparing competent cells was adopted for the majority of these experi-ments. Approximately 5 X 105 to 2 X 106 cells per ml were inoculated into NS

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