Yevtoczneric STUDIES OF MAIZE AND NEUROSPORA. Barsara McCiintock InpuctION oF MutaTIONs IN THE SHORT ARM OF CHROMOSOME 9 IN Maize In the past, many methods have been used to induce mutations. The majority of these methods do not give rise to spe- cific mutations orto mutations confined to specific regions of the chromosome com- plement. Instead, a random assortment and distribution of mutations are obtained. A better understanding of the factors in- volved in the mutation processes would be possible if specific mutations associated with specific regions of the chromosomal complement could be effected. Recent in- vestigations with maize have suggested sev- eral approaches to the problem of induc- tion of specific mutations. One of these will be considered in this report. In pre- vious reports, the repeated induction of the mutants pyd (pale-yellow seedling), wd (white seedling), and yg (yellow- green seedling and plant) has been described. Their origin has been asso- ciated with the behavior in several succes- sive nuclear divisions of a recently broken end of a chromosome. This behavior has been called the chromatid type of break- age-fusion-bridge cycle. The pyd mutant appeared when the chromosomal comple- ment was deficient for a small terminal segment of the short arm of chromosome g; the wd mutant appeared when a slightly longer terminal segment was missing. The mutant phenotype bz (bronze) has like- wise appeared following the production of a specific internal deficiency, as previously described. From this and other types of evidence, it has been concluded that spe- cific mutations will arise as the conse- quence of specific minute deficiencies. If the breakage-fusion-bridge cycle could give rise to a number of different internal minute deficiencies, and if the short arm of chromosome g were subjected to this process, various new mutants other than pyd, wd, yg, and bz should appear, each related to loss of a specific minute segment within this arm. The methods used to iso- late the mutants pyd, wd, yg, and bz were selective. Therefore, a random sample of mutants which might be produced as the consequence of the breakage-fusion-bridge cycle did not appear. During the past year, nonselective methods have been used to determine whether the expected new mu- tants actually are being produced. Cytological observations of the breakage- fusion-bridge cycle, as well as theoretical considerations, have indicated that this cycle will result in the production of in- ternal deficiencies. Occasionally, a chro- matid bridge in an anaphase figure is broken at more than one place. If a chromatid bridge breaks in three places, two centric chromosomes with a single broken end and two acentric fragments, each with both ends broken, will be formed. It is possible for the two frag- ments to enter one telophase nucleus along with the centric chromosome. If, in this nucleus, a particular type of fusion of broken ends occurs, a centric rod chromo- some with an internal deficiency and an acentric ring fragment can be produced (following fusion of the two broken ends of the proximal fragment to form an acentric ring, and fusion of one broken end of the distal fragment with the broken DEPARTMENT OF GENETICS end of the centric chromosome). If the remaining free broken end of the centric rod chromosome healed and no longer underwent the breakage-fusion-bridge cy- cle, a chromosome with an internal de- ficiency might subsequently be isolated. Sufficient cytological evidence has accumu- lated to support the assumption that this is one method of origin of internal de- ficiencies. Theoretical considerations sug- gest a second method for obtaining in- ternal deficiencies. Many investigators have considered the anaphase chromo- somes to be multiple, that is, composed of two or more. sister strands. It is prob- able that effective doubleness at anaphase is present in some cells or tissues and not in others. Should a chromatid bridge at anaphase be composed of two sister strands, breakage need not occur at comparable positions in the two strands. Should the breakage be unequal, the chromatin com- position of the two sister strands enter- ing a nucleus would not be comparable. They could differ by various duplications or deficiencies. If, in the following telo- phase, fusion occurred between the two broken ends of the unequal strands, the chromatin components between the two centromeres would consist of two dis- similar instead of similar segments. A chromatid bridge and breakage of this bridge would follow in the next mitotic division. Should the resulting newly broken end heal permanently, it might be possible subsequently to isolate a chro- matid with an internal deficiency. The type and extent of deficiency would de- pend on the positions of breakage in these two divisions. This process would give rise to internal deficiencies without frag- ment formation. Again, theoretical consid- erations have suggested that the chromo- some type of breakage-fusion-bridge cycle (see previous reports) should result in chromosomes with internal deficiencies 109 ranging from minute to extensive. There- fore, both the chromatid and the chromo- some type of breakage cycle have been utilized in an attempt to produce and isolate new mutations confined within the short arm of chromosome 9. To isolate new mutants produced by the chromatid bridge cycle, Fz progeny de- rived from F: plants that had received a recently broken chromosome 9 from one parent were examined. To isolate new mutants produced by the chromosome bridge cycle, the selfed progeny of indi- viduals that had received a newly broken chromosome g from each parent were ex- amined. In many cases, the constitution of the short arm of the chromosomes 9 with healed broken ends had been considerably altered during the period of the breakage cycles. Large as well as small duplications or deficiencies frequently were present. Many of these altered chromosomes 9 did not pass through the gametes to the next generation. Whenever the pollen grains and eggs carrying the chromo- somes g with altered short arms were capable of effecting fertilization, the selfed progeny could include individuals homo- zygous for these altered short arms. Should an alteration, when homozygous, result in a changed phenotype, individuals with a distinct mutant character would appear in the progeny. Considerations of space and labor confined the search for new muta- tions mainly to the kernels and the seed- lings. A number of new mutants appeared in these progenies. The most clearly de- fined of these mutants were selected to determine whether or not they were lo- cated in the short arm of chromosome 9. Only 3 of the distinctly new types of mutant have been sufficiently analyzed to indicate their positions in the short arm. These are a small-kernel mutant (smk), a spotted-leaf mutant (spl), and a pale-green mutant (pg). The smk and spi mutants IIo are located in the distal third of the short arm, whereas pg is located between the mutants sk and wx. Many new pyd and wd mutants and a few new yg mutants ap- peared in these cultures. Although 69 mutants arising from newly broken chro- mosomes 9 have been tested, they represent only 7 distinct phenotypes because of the repeated occurrence of the same mutations, In the published linkage group of chromo- some 9, 7 spontaneously arising mutants have been placed in the short arm. The symbols for these are: Dt, yg, C, sh, bz, bp, and wx. The newly broken chromosomes g have given the 7 mutants pyd, wd, yg, smk, spl, bz, and pg. As has been stated previously, the yg and 4z mutants derived from the broken chromosomes g are allelic to the 2 mutants, yg and dz, that arose spontaneously in genetic cultures. An interesting type of chromosomal be- havior has appeared in three of the broken- chromosome cultures mentioned above. In each culture, one of the broken chromo- somes 9 is continually being lost from cells during development. This loss is not due to bridge formation or to ring chromosome behavior, but appears to be caused by the inability of the two halves of this chromo- some to migrate to opposite poles in some of the somatic anaphase figures. The rate of loss varies widely from plant to plant. Within a single plant, changes in rate occur; this is made evident by the pres- ence of distinct sectors each with its own rate of loss. To date, only a cursory ex- amination of the nature of this phenome- non has been made; it warrants further study. In addition, some of the mutants appearing in these cultures are individu- ally provocative. Several show variegation characterized by a change from mutant to normal-appearing tissues. For any one plant, a distinctive or basic rate of change is apparent, but this basic rate differs from plant to plant. Sectors with changed rates CARNEGIE INSTITUTION OF WASHINGTON of variegation appear in all plants, espe- cially in the later-appearing tissues. It is significant that twin sectors accompany many if not most of the alterations in rate; this is expressed by the appearance of a sector of tissue having a greatly increased rate of variegation immediately adjacent to a sector of tissue having a much reduced rate of variegation. , PRELIMINARY STUDIES OF THE CHROMOSOMES OF THE FuNcus NEUROSPORA CRASSA During the fall of 1944, a period of ten weeks was spent in the Biological Labora- tories of Stanford University, where ge- netic studies are being conducted with the fungus Neurospora. The purpose of this visit was to obtain some knowledge of chromosomal and nuclear behavior in Neurospora crassa. Although fungi have assumed an important role as genetic ma- terials, little has been done to coordinate the genetic studies with a study of chromo- somal conditions. As genetic investiga- tions with fungi progress, the necessity for correlative cytogenetic analyses will be- come increasingly evident. It was a pleas- ure to have the opportunity of examining Neurospora in this laboratory. Progress was greatly accelerated by the availability of large numbers of stocks, both wild-type and mutant, and by the generous and co- operative support of the members of the department. The observations were confined to the chromosomes and nuclei of the ascus. They included observations of chromo- some numbers, absolute and relative sizes of the chromosomes, centromere positions, internal organization of the chromosomes, zygote formation, chromosome behavior in the two meiotic mitoses and the equa- tional mitosis which follows, and scattered observations of several chromosomal trans- locations. In the short time available, no DEPARTMENT OF GENETICS one of these topics could be adequately considered. Nevertheless, this over-all sur- vey has suggested that some fungi may be adequate and, in several respects, superior material for cytogenetic studies. The haploid number of chromosomes in Neurospora crassa is 7. Each chromosome of the complement is distinguished by its relative length, the position of its centro- mere, and its internal organization. The longest chromosome is approximately 2.7 times as long as the shortest. The second- longest chromosome, chromosome 2, has a nucleolus organizer located close to the end of the short arm. The organizer region functions to produce a nucleolus in a man- ner similar to that observed in many other organisms. Because of its location close to the end of one arm of this chromosome, a minute satellite is formed. Throughout the various nuclear cycles, the relative lengths of the chromosomes of the complement are maintained. Therefore, absolute lengths need be given only for the longest chromo- some. In the third division in the ascus, which is equational, this chromosome may be only 1.5 microns long. At the full meiotic prophase extension, it may be 15 microns long. Chromomere patterns were observed at this latter stage; each chromo- some appears to have its characteristic pattern. Centromere positions were ade- quately determined for the two longest chromosomes, and approximate positions were obtained for the other five chromo- somes. ‘Two heterochromatic segments were observed and located adjacent to the centromere, but the chromosome or chro- mosomes carrying these heterochromatic segments were not identified. Fusion of two haploid nuclei to form the zygote nucleus occurs in the very young ascus. The two sets of chromo- somes in this zygote nucleus then com- mence the activities associated with meiosis. The behavior of the chromosomes in the Ilr early meiotic stages is of considerable theo- retical interest. During meiosis in most organisms, homologous associations com- mence when the chromosomes are in a very elongated state. In the Neurospora strains most intensively studied, this occurs when the chromosomes are greatly con- tracted. Following nuclear fusion, the chromosomes contributed by each nucleus undergo what appears to be a typical pro- phase contraction without visible evidence of splitting, until, in some strains, the chro- mosomes are almost as short as those of the metaphase of the third division in the ascus. In this highly contracted state, the homologous chromosomes commence their synaptic associations. Before the chromo- somes have reached this state, fusion of the nucleoli contributed by the two nuclei usu- ally has occurred. Actual physical associa- tion of the homologues usually begins at one or both ends and continues along the chromosomes. In many nuclei, synapsis is completed for some pairs of chromosomes before the members of the other pairs have approached sufficiently close to each other to commence actual contacts. It is not clear from these studies whether the approach of homologous chromosomes toward each other is directed or whether it follows from random movements of the chromosomes in the nucleus. It is of con- siderable theoretical interest to determine the range of the synaptic force which brings about homologous associations of chromosomes. It is suspected that the young asci of Neurospora might be readily cultured. Because of the relatively large volume of the nucleus and the small size of the chromosomes in these asci, continuous observations of the behavior of these chro- mosomes in the living nuclei might be possible. Following the synaptic phase, the asso- ciated homologous chromosomes begin to elongate until, as stated above, the longest 112 chromosome may reach a length of 15 microns. Diplotene sets in rather suddenly following the completion of elongation of the synapsed chromosomes. The period from diplotene to metaphase I is passed through very rapidly. At diakinesis, typical chiasmata may be observed leading to rather orthodox, even though small, meta- phase I bivalents. Although the nucleolus becomes smaller during the prometaphase stage, it is still present at metaphase. Chro- mosome 2 remains attached to the nucleo- lus by its organizer region. Anaphase I appears to be essentially typical except for the presence of the nucleolus. The nucleo- lus may be dragged toward one pole or stretched between the poles because the nucleolus organizer of one or more chro- matids of chromosome 2 still remains at- tached to it. The nucleolus becomes de- tached before telophase sets in. At telo- phase I, and likewise at telophases II and III, the centromere regions of all the chro- mosomes form an aggregate that lies at the apex of a distinct protrusion of the nucleus (the beak). No true resting nu- cleus is formed. Instead, the chromosomes uncoil, the individual arms of each chro- mosome extending into an elongated nu- cleus. A new nucleolus is formed and remains attached to the nucleolus organ- izers of chromosome 2. Contraction of the chromosomes initiates prophase II. This continues until the two dyad chromosomes are in the form of short, parallel rods, each showing a conspicuous centromere region. Metaphase and anaphase II are essentially typical. At telophase ‘II the centromere re- gions are again aggregated at the apex of the beak of the nucleus; the chromo- somes uncoil and the two arms of each CARNEGIE INSTITUTION OF WASHINGTON chromosome extend into the nucleus as individual strands. They remain in this condition until the following prophase. The extent of elongation of the chromo- somes appears to be similar to that ob- served in the meiotic prophase. In each nucleus, a new nucleolus is formed at the position of the nucleolus organizers of chromosome 2. Prophase III is initiated by contraction of the arms of the chromo- somes. The metaphase and anaphase of division III proceed as a typical equational mitosis. The resting stage of nuclear or- ganization follows telophase III. Shortly after spore delimitation, a mitosis occurs in each ascus. This is also a typical equa- tional mitosis. In essential details, divi- sions I and II are typically meiotic. Divi- sion III is essentially a somatic mitosis, ex- cept that the chromosomes retain their identity as elongated strands from the telo- phase of division II to the prophase of division III. The time of effective splitting of the chromosomes for this division is of some theoretical interest. Because many of the mutations in New- rospora have appeared following X-ray and ultraviolet irradiation, it was suspected that various types of chromosomal translo- cation might likewise have been induced by these treatments. Three irradiation-in- duced mutants, whose genetic behavior suggested the presence of some chromo- somal abnormality, were selected for ex- amination. A translocation between two nonhomologous chromosomes was found in each case. Intensive studies of these translocations were not undertaken, but the preliminary observations have sug- gested the usefulness of some transloca- tions for attacking special problems.