Inheritance of histone H3 variants across mitotic cell divisions

Dissertation presented to obtain the Ph.D degree in Molecular Biology.

Cohesion decay: quantitative analysis of partial sister chromatid cohesion

Cell division is a highly dynamic process where sister chromatids remain associated with each other from the moment of DNA replication until the later stages of mitosis, giving rise to two daughter cells with equal genomes. The “molecular glue” that links sister DNA molecules is called cohesin, a tripartite ring-like protein complex composed of two Structural Maintenance of Chromosome proteins (Smc1 and Smc3) bridged by a kleisin subunit Rad21/Scc1, that together prevent precocious sister chromatid separation. Accumulating evidence has suggested that cohesion decay may be the cause of segregation errors that underlie certain human pathologies. However it remains to be determined how much cohesin loss abolishes functional sister chromatid cohesion. To answer these questions, we have developed different experimental conditions aiming to titrate the levels of cohesin on mitotic chromosomes in a precise manner. Using these tools, we will determine the minimal amount of cohesin needed to confer functional cohesion. The approaches described here take advantage of a system in Drosophila melanogaster where the Tobacco Etch Virus (TEV) protease can cleave the Rad21 subunit of cohesin leading to precocious sister chromatid separation. Firstly, we tried to express different levels of TEV protease to obtain partial loss of cohesion. However, this approach has failed to produce systematic different levels of sister chromatid separation. Most of the work was therefore focused on a second strategy, for which we established strains with different levels of cohesin sensitive/cohesin resistant to TEV protease. Strains containing different amounts of functional cohesin (TEV resistant) were tested by in vitro cleavage and by in vivo injections in embryos for their ability to promote sister chromatid cohesion. Our results reveal that removal of half of the cohesin complexes does not impair chromosome segregation, implying that chromosome cohesion is less sensitive to cohesin amounts than previously anticipated.

Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His, R337H) founder mutation : clues from haplotyping of short tandem repeats on Chromosome 17p

Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial his- tory. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Ibe- ric origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenera- tional distance) and thus pre-dating European migration to Brazil. So far, the founder p. Arg337His haplotype has not been detected outside Brazil, with the exception of two resi- dents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.

Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

Background: To alert for the diagnosis of the 22q11.2 deletion syndrome (22q11.2DS) in patients with congenital heart disease (CHD). Objective: To describe the main CHDs, as well as phenotypic, metabolic and immunological findings in a series of 60 patients diagnosed with 22q11.2DS. Methods: The study included 60 patients with 22q11.2DS evaluated between 2007 and 2013 (M:F=1.3, age range 14 days to 20 years and 3 months) at a pediatric reference center for primary immunodeficiencies. The diagnosis was established by detection of the 22q11.2 microdeletion using FISH (n = 18) and/or MLPA (n = 42), in association with clinical and laboratory information. Associated CHDs, progression of phenotypic facial features, hypocalcemia and immunological changes were analyzed. Results: CHDs were detected in 77% of the patients and the most frequent type was tetralogy of Fallot (38.3%). Surgical correction of CHD was performed in 34 patients. Craniofacial dysmorphisms were detected in 41 patients: elongated face (60%) and/or elongated nose (53.3%), narrow palpebral fissure (50%), dysplastic, overfolded ears (48.3%), thin lips (41.6%), elongated fingers (38.3%) and short stature (36.6%). Hypocalcemia was detected in 64.2% and decreased parathyroid hormone (PTH) level in 25.9%. Decrease in total lymphocytes, CD4 and CD8 counts were present in 40%, 53.3% and 33.3%, respectively. Hypogammaglobulinemia was detected in one patient and decreased concentrations of immunoglobulin M (IgM) in two other patients. Conclusion: Suspicion for 22q11.2DS should be raised in all patients with CHD associated with hypocalcemia and/or facial dysmorphisms, considering that many of these changes may evolve with age. The 22q11.2 microdeletion should be confirmed by molecular testing in all patients.

Hereditariedade da homeose

A preliminary account on the normal development of the imaginai discs in holometabolic Insects is made to serve as an introduction to the study of the hereditary homoeosis. Several facts and experimental data furnished specially by the students of Drosophila are brought here in searching for a more adequate explanation of this highly interesting phenomenon. The results obtained from the investigations of different homoeotic mutants are analysed in order to test Goldschmidt's theory of homoeosis. Critical examination of the basis on which this theory was elaborated are equally made. As a result from an extensive theoretical consideration of the matter and a long discussion of the most recent papers on this subject the present writer concludes that the Goldschmidt explanation of the homoeotic phenomena based on the action of diffusing substances produced by the genes, the "evocators", and on the alteration of the normal speed of maturation of the imaginai discs equally due to the activity of the genes, could not be proved and therefore should be abandoned. In the same situation is any other explanation like that of Waddington or Villee considered as fundamentally identical to that of Goldschmidt. In order to clear the problem of homoeosis in terms which seem to put the phenomenon in complete agreement with the known facts the present writer elaborated a theory first published a few years ago (1941) based entirely on the assumption that the imaginai discs are specifically determined by some kind of substances, probably of chemical nature, contained in the cytoplam of the cells entering in the consti- tution of each individual disc. These substances already present in the blastem of the egg in which they are distributed in a definite order, pass to different cells at the time the blastem is transformed into blastoderm. These substances according to their organogenic potentiality may be called antenal-substance, legsubstance, wing-substance, eye-substance, etc. The hipoderm of the embryo resulting from the multiplication of the blastoderm cells would be constituted by a series of cellular areas differing from each other in their particular organoformative capacity. Thus the hypoderm giving rise to the imaginai discs, it follows that each disc must have the same organogenic power of the hypodermal area it came from. Therefore the discs i*re determinated since their origin by substances enclosed in the cytoplasm of their cells and consequently can no longer alter their potentiality. When an antennal disc develops into a leg one can conclude that this disc in spite of its position in the body of the larva is not, properly speaking, an antennal disc but a true leg disc whose cells instead of having in their cytoplasm the antennal substance derived from the egg blastem have in its place the leg-substance. Now, if a disc produces a tarsus or an antenna or even a compound appendage partly tarsus-like, partly antenna-like, it follows tha,t both tarsal and antennal substances are present in it. The ultimate aspect of the compound structure depends upon the reaction of each kind of substance to the different causes influencing development. For instance, temperature may orient the direction of development either lowards arista or tarsus, stimulating, or opposing to the one or the other of these substances. Confering to the genes the faculty of altering the constitution of the substances containing in the cytoplasm forming the egg blastem or causing transposition of these substances from one area to another or promoting the substitution of a given substance by a different one, the hereditary homoeocis may be easily explained. However, in the opinion of the present writer cytoplasm takes the initiative in all developmental process, provoking the chromosomes to react specifically and proportionally. Accordingly, the mutations causing homoeotic phenomena may arise independently at different rime in the cytoplasm and in the chromosomes. To the part taken by the chromosomes in the manifestation of the homoeotic characters is due the mendalian ratio observed in homoeotic X normal crosses. Expression, in itself, is mainly due to the proportion of the different substances in the cells of the affected discs. Homoeotic phenomena not presenting mendelian ratio may appear as consequence of cytoplasmic mutation not accompanied by chromosomal mutation. The great variability in the morphology of the homoeotic characteres, some individual being changed towards an extreme expression of the mutant phenotype while others in spite of their homozigous constitution cannot be distinguished from the normal ones, strongly supports the interpretation based on the relative proportion of the determining substances in the discs. To the same interpretation point also asymetry and other particularities observed in the exteriorization of the phenomenon. In conformity with this new conception homoeosis should not prove homology of Insect appendages (Villee 1942) since a more replacement of substances may cause legs to develop in substitution of the wings, as it was already observed (requiring confirmation in the opinion of Bateson 1894, p. 184) and no one would conclude for the homology of these organs in the usual meaning of the term.

Estudos citológicos em Hemíoteros da família Coreidae

A more or less detailed study of the spermatogenesis in six species of Hemiptera belonging to the Coreid Family is made in the present paper. The species studied and their respective chromosome numbers were: 1) Diactor bilineatus (Fabr.) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationaliv in the first division and passing undivided to one pole in the second. 2) Lcptoglossus gonagra (Fabr.) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationally in the first division and passing undivided to one pole in the second. 3) Phthia picta (Drury) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationally in the first division and passing undivided to one pole in the second. 4) Anisocelis foliacea Fabr. : spermatogonia with 26 + X fthe highest mumber hitherto known in the Family), primary .spermatocytes with 13 + X, X dividing equationally in the first division an passing undivided to one pole in the second. 5) Pachylis pharaonis (Herbtst) : spermatogonia with 16 + X, primary spermatocytes with 8 + X. Behaviour of the heteroehromosome not referred. 6) Pachylis laticornis (Fabr.) : spermatogonia with 14 + X, primary spermatocytes with 7 + X, X passing undivided to one pole in the first division and therefore secondary spermatocytes with 7 + X and 7 chromosomes. General results and conclusions a) Pairing modus of the chromosomes (Telosynapsis or Farasynapsis ?) - In several species of the Coreld bugs the history of the chromosomes from the diffuse stage till diakinesis cannot be follewed in detail due specially to the fact that lhe bivalents, as soon as they begin to be individually distinct they appear as irregular and extremely lax chromatic areas, which through an obscure process give rise to the diakinesis and then to the metaphase chomosomes. Fortunately I was able to analyse the genesis of the cross-shaped chromosomes, becoming thus convinced that even in the less favorable cases like that of Phthia, in which the crosses develop from four small condensation areas of the diffuse chromosomes, nothing in the process permit to interpret the final results as being due to a previous telosynaptic pairing. In the case of long bivalents formed by two parallel strands intimately united at both endsegments and more or less widely open in the middle (Leptoglossus, Pachylis), I could see that the lateral arms of the crosses originate from condensation centers created by a torsion or bending in the unpaired parts of the chromosomes In the relatively short bivalents the lateral branches of the cross are formed in the middle but in the long ones, whose median opening is sometimes considerable, two asymetrical branches or even two independent crosses may develop in the same pair. These observations put away the idea of an end-to-end pairing of the chromosomes, since if it had occured the lateral arms of the crosses would always be symetrical and median and never more than two. The direct observation of a side- toside pairing of the chromosomal threads at synizesis, is in foil agreement with the complete lack of evidence in favour of telosynapsis. b) Anaphasic bridges and interzonal connections - The chromosomes as they separate from each other in anaphase they remain connected by means of two lateral strands corresponding to the unpaired segmenas observed in the bivalents at the stages preceding metaphase. In the early anaphase the chromosomes again reproduce the form they had in late diafcinesis. The connecting threads which may be thick and intensely coloured are generally curved and sometimes unequal in lenght, one being much longer than the other and forming a loop outwardly. This fact points to a continuous flow of chromosomal substance independently from both chromosomes of the pair rather than to a mechanical stretching of a sticky substance. At the end of anaphase almost all the material which formed the bridges is reduced to two small cones from whose vertices a very fine and pale fibril takes its origin. The interzonal fibres, therefore, may be considered as the remnant of the anaphasic bridges. Abnormal behaviour of the anaphase chromosomes showed to be useful in aiding the interpretation of normal aspects. It has been suggested by Schrader (1944) "that the interzonal is nothing more than a sticky coating of the chromosome which is stretched like mucilage between the daughter chromosomes as they move further and further apart". The paired chromosomes being enclosed in a commom sheath, as they separate they give origin to a tube which becomes more and more stretched. Later the walls of the tube collapse forming in this manner an interzonal element. My observations, however, do not confirm Schrader's tubular theory of interzonal connections. In the aspects seen at anaphase of the primary spermatocytes and described in this paper as chromosomal bridges nothing suggests a tubular structure. There is no doubt that the chromosomes are here connected by two independent strands in the first division of the spermatocytes and by a single one in the second. The manner in which the chromosomes separate supports the idea of transverse divion, leaving little place for another interpretation. c) Ptafanoeomc and chromatoid bodies - The colourabtlity of the plasmosome in Diactor and Anisocelis showed to be highly variable. In the latter species, one may find in the same cyst nuclei provided with two intensely coloured bodies, the larger of which being the plasmosome, sided by those in which only the heterochromosome took the colour. In the former one the plasmosome strongly coloured seen in the primary metaphase may easily be taken for a supernumerary chromosome. At anaphase this body stays motionless in the equator of the cell while the chromosomes are moving toward the poles. There, when intensely coloured ,it may be confused with the heterochromosome of the secondary spermatocytes, which frequently occupies identical position in the corresponding phase, thus causing missinterpretation. In its place the plasmosome may divide into two equal parts or pass undivided to one cell in whose cytoplasm it breaks down giving rise to a few corpuscles of unequal sizes. In Pachylis pharaonis, as soon as the nuclear membrane breate down, the plasmosome migrates to a place in the periphery of the cell (primary spermatocyte), forming there a large chromatoid body. This body is never found in the cytoplasm prior to the dissolution of the nuclear membrane. It is certain that chromatoid bodies of different origin do exist. Here, however, we are dealing, undoubtedly, with true plasmosomes. d) Movement of the heterochromosome - The heterochromosome in the metaphase of the secondary spermatocytes may occupy the most different places. At the time the autosomes prient themselves in the equatorial plane it may be found some distance apart in this plane or in any other plane and even in the subpolar and polar regions. It remains in its place during anaphase. Therefore, it may appear at the same level with the components of one of the anaphase plates (synchronism), between both plates (succession) or between one plate and tbe pole (precession), what depends upon the moment the cell was fixed. This does not mean that the heterochromosome sometimes moves as quickly as the autosomes, sometimes more rapidly and sometimes less. It implies, on the contrary, that, being anywhere in the cell, the heterochromosome m he attained and passed by the autosomes. In spite of being almost motionless the heterochromosome finishes by being enclosed in one of the resulting nuclei. Consequently, it does move rapidly toward the group formed by the autosomes a little before anaphase is ended. This may be understood assuming that the heterochromosome, which do not divide, having almost inactive kinetochore cannot orient itself, giving from wherever it stays, only a weak response to the polar influences. When in the equator it probably do not perform any movement in virtue of receiving equal solicitation from both poles. When in any other plane, despite the greater influence of the nearer pole, the influence of the opposite pole would permit only so a slow movement that the autosomes would soon reach it and then leave it behind. It is only when the cell begins to divide that the heterochromosome, passing to one of the daughter cells scapes the influence of the other and thence goes quickly to join the autosomes, being enclosed with them in the nucleus formed there. The exceptions observed by BORING (1907) together with ; the facts described here must represent the normal behavior of the heterocromosome of the Hemiptera, the greater frequency of succession being the consequence of the more frequent localization of the heterochromosome in the equatorial plane or in its near and of the anaphase rapidity. Due to its position in metaphase the heterochromosome in early anaphase may be found in precession. In late anaphase, oh the contrary ,it appears almost always in succession. This is attributed to the fact of the heterochromosome being ordinairily localized outside the spindle area it leaves the way free to the anaphasic plate moving toward the pole. Moreover, the heterochromosome being a round element approximately of the size of the autosomes, which are equally round or a little longer in the direction of the movement, it can be passed by the autosomes even when it stands in the area of the spindle, specially if it is not too far from the equatorial plane. e) The kinetochore - This question has been fully discussed in another paper (PIZA 1943a). The facts treated here point to the conclusion that the chromosomes of the Coreidae, like those of Tityus bahiensis, are provided with a kinetochore at each end, as was already admitted by the present writer with regard to the heterochromosome of Protenor. Indeed, taking ipr granted the facts presented in this paper, other cannot be the interpretation. However, the reasons by which the chromosomes of the species studied here do not orient themselves at metaphase of the first division in the same way as the heterochromosome of Protenor, that is, with the major axis parallelly to the equatorial plane, are claiming for explanation. But, admiting that the proximity of the kinetochores at the ends of chromosomes which do not separate until the second division making them respond to the poles as if they were a single kinetochore ,the explanation follows. (See PIZA 1943a). The median opening of the diplonemas when they are going to the diffuse stage as well as the reappearance of the bivalents always united at the end-segments and open in the middle is in full agreement with the existence of two terminal kinetochores. The same can be said with regard to the bivalents which join their extremities to form a ring.

Comportamento do Heterocromossômio em alguns Ortópteros do Brasil

In the present paper the behavior of the heterochromoso-mes in the course of the meiotic divisions of the spermatocytes in 15 species of Orthoptera belonging to 6 different families was studied. The species treated and their respective chromosome numbers were: Phaneropteridae: Anaulacomera sp. - 1 - 2n = 30 + X, n +15+ X and 15. Anaulacomera sp. - 2 - 2n - 30 + X, n = 15+ X and 15. Stilpnochlora marginella - 2n = 30 + X, n = 15= X and 15. Scudderia sp. - 2n = 30 + X, n = 15+ X and 15. Posldippus citrifolius - 2n = 24 + X, n = 12+X and 12. Acrididae: Osmilia violacea - 2n = 22+X, n = 11 + X and 11. Tropinotus discoideus - 2n = 22+ X, n = 11 + X and 11. Leptysma dorsalis - 2n = 22 + X, n = 11-J-X and 11. Orphulella punctata - 2n = 22-f X, n = 11 + X and 11. Conocephalidae: Conocephalus sp. - 2n = 32 + X, n = 16 + X and 16. Proscopiidae: Cephalocoema zilkari - 2n = 16 + X, n = 8+ X and 8. Tetanorhynchus mendesi - 2n = 16 + X, n = 8+X and 8. Gryliidae: Gryllus assimilis - 2n = 28 + X, n = 14+X and 14. Gryllodes sp. - 2n = 20 + X, n = 10- + and 10. Phalangopsitidae: Endecous cavernicola - 2n = 18 +X, n = 94-X and 9. It was pointed out by the present writer that in the Orthoptera similarly to what he observed in the Hemiptera the heterochromosome in the heterocinetic division shows in the same individual indifferently precession, synchronism or succession. This lack of specificity is therefore pointed here as constituting the rule and not the exception as formerly beleaved by the students of this problem, since it occurs in all the species referred to in the present paper and probably also m those hitherto investigated. The variability in the behavior of the heterochromosome which can have any position with regard to the autosomes even in the same follicle is attributed to the fact that being rather a stationary body it retains in anaphase the place it had in metaphase. When this place is in the equator of the cell the heterochromosome will be left behind as soon as anaphase begins (succession). When, on the contrary, laying out of this plane as generally happens (precession) it will sooner be reached (synchronism) or passed by the autosomes (succession). Due to the less kinetic activity of the heterochromosome it does not orient itself at metaphase remaining where it stands with the kinetochore looking indifferently to any direction. At the end of anaphase and sometimes earlier the heterochromosome begins to show mitotic activities revealed by the division of its body. Then, responding to the influence of the nearer pole it moves to it being enclosed with the autosomes in the nucleus formed there. The position of the heterochromosome in the cell is explained in the following manner: It is well known that the heterochromosome of the Orthoptera is always at the periphery of the nucleus, just beneath the nuclear membrane. This position may be any in regard of the axis of the dividing cell, so that if one of the poles of the spindle comes to coincide with it, the heterochromosome will appear at this pole in the metaphasic figures. If, on the other hand, the angle formed by the axis of the spindle with the ray reaching the heterochromosome increases the latter will appear in planes farther and farther apart from the nearer pole until it finishes by being in the equatorial plane. In this way it is not difficult to understand precession, synchronism or succession. In the species in which the heterochromosome is very large as it generally happens in the Phaneropteridae, the positions corresponding to precession are much more frequent. This is due to the fact that the probabilities for the heterochromosome taking an intermediary position between the equator and the poles at the time the spindle is set up are much greater than otherwise. Moreover, standing always outside the spindle area it searches for a place exactly where this area is larger, that is, in the vicinity of the poles. If it comes to enter the spindle area, what has very little probability, it would be, in virtue of its size, propelled toward the pole by the nearing anaphasic plate. The cases of succession are justly those in which the heterochromosome taking a position parallelly to the spindle axis it can adjust its large body also in the equator or in its proximity. In the species provided with small heterochromosome (Gryllidae, Conocephalidae, Acrididae) succession is found much more frequently because here as in the Hemiptera (PIZA 1945) the heterochromosome can equally take equatorial or subequatorial positions, and, furthermore, when in the spindle area it does offer no sereous obstacle to the passage of the autosomes. The position of the heterochromosome at the periphery of the nucleus at different stages may be as I suppose, at least in part a question of density. The less colourability and the surface irregularities characteristic of this element may well correspond to a less degree of condensation which may influence passive movements. In one of the species studied here (Anaulacomera sp.- 1) included in the Phaneropteridae it was observed that the plasmosome is left motionless in the spindle as the autosomes move toward the poles. It passes to one of the secondary spermatocytes being not included in its nucleus. In the second division it again passes to one of the cells being cast off when the spermatid is being transformed into spermatozoon. Thus it is regularly found among the tails of the spermatozoa in different stages of development. In the opinion of the present writer, at least in some cases, corpuscles described as Golgi body's remanents are nothing more than discarded plasmosomes.

Estudos genéticos sôbre o milho tunicata

The study of pod corn seems still of much importance from different points of view. The phylogenetical importance of the tunicate factor as a wild type relic gene has been recently discussed in much detail by MANGELSDORF and REEVES (1939), and by BRIEGER (1943, 1944a e b). Selection experiments have shown that the pleiotropic effect of the Tu factor can be modified very extensively (BRIEGER 1944a) and some of the forms thus obtained permitt comparison of male and female inflorescences in corn and related grasses. A detailed discussion of the botanical aspect shall be given shortly. The genetic apect, finally, is the subject of the present publication. Pod corn has been obtained twice: São Paulo Pod Corn and Bolivia Pod Corn. The former came from one half ear left in our laboratory by a student and belongs to the type of corn cultivated in the State of São Paulo, while the other belongs to the Andean group, and has been received both through Dr. CARDENAS, President of the University at Cochabamba, Bolivia, and through Dr. H. C. CUTLER, Harvard University, who collected material in the Andes. The results of the studies may be summarized as follows: 1) In both cases, pod corn is characterized by the presence of a dominant Tu factor, localized in the fourth chromosome and linked with sul. The crossover value differs somewhat from the mean value of 29% given by EMERSON, BEADLE and FRAZER (1935) and was 25% in 1217 plants for São Paulo Pod Corn and 36,5% in 345 plants for Bolivia Pod Corn. However not much importance should be attributed to the quantitative differences. 2) Segregation was completely normal in Bolivia Pod Corn while São Paulo Pod Corn proved to be heterozygous for a new com uma eliminação forte, funcionam apenas 8% em vez de 50%. Existem cerca de 30% de "jcrossing-over entre o gen doce (Su/su) e o fator gametofítico; è cerca de 5% entre o gen Tu e o fator gametofítico. A ordem dos gens no cromosômio IV é: Ga4 - Tu - Sul. 3) Using BRIEGER'S formulas (1930, 1937a, 1937b) the following determinations were made. a) the elimination of ga4 pollen tubes may be strong or weak. In the former case only about 8% and in the latter 37% of ga4 pollen tubes function, instead of the 50% expected in normal heterozygotes. b) There is about 30,4% crossing-over between sul and ga4 and 5,3% between Tu and ga3, the order of the factors beeing Su 1 - Tu - Ga4. 4) The new gametophyte factor differs from the two others factors in the same chromosome, causing competition between pollen tubes. The factor Gal, ocupies another locus, considerably to the left of Sul (EMERSON, BEADLE AND FRAZSER, 1935). The gen spl ocupies another locus and causes a difference of the size of the pollen grains, besides an elimination of pollen tubes, while no such differences were observed in the case of the new factor Ga4. 5) It may be mentioned, without entering into a detailed discussion, that it seems remarquable that three of the few gametophyte factors, so far studied in detail are localized in chromosome four. Actuality there are a few more known (BRIEGER, TIDBURY AND TSENG 1938), but only one other has been localized so far, Ga2, in chromosome five between btl and prl. (BRIEGER, 1935). 6) The fourth chromosome of corn seems to contain other pecularities still. MANGELSDORF AND REEVES (1939) concluded that it carries two translocations from Tripsacum chromosomes, and BRIEGER (1944b) suggested that the tu allel may have been introduced from a tripsacoid ancestor in substitution of the wild type gene Tu at the beginning of domestication. Serious disturbances in the segregation of fourth chromosome factors have been observed (BRIEGER, unpublished) in the hybrids of Brazilian corn and Mexican teosinte, caused by gametophytic and possibly zygotic elimination. Future studies must show wether there is any relation between the frequency of factors, causing gametophyte elimination and the presence of regions of chromosomes, tranfered either from Tripsacum or a related species, by translocation or crossing-over.

Notas sôbre a meiose de pachylis (Hemipt-Coreidae)

Spermatogonial chromosomes of Pachylis laticornis and Pachylis pharaonis begin anaphasic movement with both ends turned toward the same pole, maintaining this form util they reach the poles. This is a proof that they are provided with one kinetochore at each end. Additional proof for a longitudinal division of each longitudinal half of the anaphase chromosomes of the primary sper- matocytes is presented against the idea of a previous end-toend pairing at metaphase. The longitudinal split of the chromosomes of the secondary spermatocytes which used to be considered as tertiary split is therefore a true secondary split. The heterochromosome in both species passes undivided to one pole in the first division of the spermatocyte. In Pachylis laticornis it appears connected with the poles by means of two fibrils detached from each extremity, what may be considered as indicating a rather premature longitudinal spliting. The behavior of the heterochromosome of Pachylis pharaonis is highly interesting and affords one of the most beautiful evidences in favour of the dicentricity of the chromosomes. Really, in metaphase the heterochromosome appears at the equator of the cell with a more or less round shape. In the beginning of anaphase it becomes fusiform. As anaphase proceeds it distends itself between the autosomal plates forming a long fusiform bridge or sends toward the plates a thick chromosomal thread. The bulky part of the heterochromosome as it passes to one side it reincorporates the substance of the thread in this side. The thread in the other side, which becomes generally thiner, is left with its kinetochore in the cell at this side. The heterochromosome therefore becomes terminally monocentric in the first division of the spermatocyte. Some figures, however, suggest that the heterochromossome from time to time may pass with both kinetochores to one of the cells, as ordinarily happens in the case of Pachylis laticornis. Summing up, other things apart the behavior of the heterochromosome in both species studied here puts out of doubt the question of the existence of two terminally located kinetochores.

Uma nova modalidade de sexo-determinação no grilo sul-americano eneoptera surinamensis

The male of Eneoptera surinamensis (Orthoptera-Eneopteridae) is provided with 9 chromosomes, that is, with 3 pairs of autosomes and 3 sex chromosomes. Spermatogonia. - The autosomes of the spermatogonia are of the same size and U-shaped. One of the sex chromosomes approximately equalling the autosomes in size is telocentric, while the other two are much larger and V-shaped. One of the latter is smaller than the other. The sex chromosomes as showed in Figs. 1 and 2 are designated by X, Yl and Y2, X being the larger V, Yl the smaller one and Y2 the rod-shaped. Primary spermatocytes. - Before the growth period of the spermatocytes all the three sex chromosomes are visible in a state of strong heteropycnosis. X is remarkable in this stage in having two long arms well separated by a wide commissural segment. (Figs. 4, 5 and 6). During the growth period Y2 disappears, while X and Yl remain in a condensed form until metaphase. These may be separated from one another or united in the most varied and irregular manner. (Fig. 7 to 12). In the latter case the segments in contact seem to be always different so that we cannot recognize any homology of parts in the sense os genetics. At diplotene Y2 reappears together with the autosomal tetrads. X and Yl may again be seen as separate or united elements. (Figs. 13 and 14). At later diakinesis and metaphase the three sex chromosomes are always independent from each other, Y2 being typically rod-shaped, X and Yl V-shaped, X being a little larger than Yl. (Fig. 15 to 18). At metaphase the three condensed tetrads go to the equatorial plane, while the sex chromosomes occupy any position at both sides of this plane. In almost all figures which could be perfectly analysed X appeared at one side of the autosomal plate an Yl together with Y2 far apart at the other side. (Figs. 16 and 18). Only a few exception have been found. (Figs. 17 and 19). At anaphase X goes in precession to one pole, Yl and Y2 to the other (Figs. 20 and 21). As it is suggested by the few figures in which a localization of the sex chromosomes different from the normal has been observed, the possibility of other types of segregation of these elements cannot be entirely precluded. But, if this does happen, the resulting gametes should be inviable or give inviable zygotes. Early in anaphase autosomes and sex chromosomes divide longitudinally, being maintained united only by the kinetochore. (Figs. 20 and 21). At metaphase the three sex chromosomes seem to show no special repulsion against each other, X being found in the proximity of Yl or Y2 indifferently. At anaphase, however, the evidences in hand point to a stronger repulsion between X on the one side and both Ys on the other, so that in spite of the mutual repulsion of the latter they finish by going to the same pole. Secondary spermatocytes. - At telophase of the primary spermatocytes all the chromosomes enter into distension without disappearing of view. A nuclear membrane is formed around the chromosomes. All the chromosomes excepting Y2 which has two arms, are four-branched. (Fig. 22). Soon the chromosomes enter again into contraction giving rise to the secondary metaphase plate. Secondary spermatocytes provided as expected with four and five chromosomes are abundantly found. (Figs. 23 and 24). In the former all chromosomes are X-shaped while in the latter there is one which is V-shaped. This is the rod- shaped Y2. In the anaphase of the spermatocytes with four chromosomes all the chromosomes are V-shaped, one of them (X) being much larger than the others. In those with five there is one rod-shaped chromosome (Y2). (Fig. 25), Spermatids. Two classes of spermatids are produced, one with X and other with Yl and Y2. All the autosomes as well as Y2 soon enter into solution, X remaining visible for long time in one class and Yl in the other. (Figs. 26 and 27). Since both are very alike at this stage, one cannot distinguish the two classes of spermatids. Somatic chromosomes in the famale. - In the follicular cells of the ovary 8 chromosomes were found, two of which are much larger than the rest. (Figs. 29 and 30). These are considered as being sex chromosomes. CONCLUSION: Eneoptera surinamensis has a new type of sex-determining mechanism, the male being X Yl Y2 and the female XX. The sex chromosomes segregate without entering into contact at metaphase or forming group. After a review of the other known cases of complex sex chromosome mechanism the author held that Eneoptera is the unique representative of a true determinate segregation of sex chromosomes. Y2 behaving as sex chromosome and as autosome is considered as representing an intermediary state of the evolution of the sex chromosomes.

Provas adicionais da dicentricidade dos cromossômios dos Hemípteros

In order to test Piza's conclusions regarding the dicentricity of Hemipteran chromosomes, two species of bugs of the family Coreidae, namely, Anasa sp. and Leptoglossus stigma (Herbst), are studied in the present paper. a) Anasa sp. - The male of this species has 21 chromosomes, that is, 20 pairs of autosomes and a single sex chromosome. The latter divides equationally in the first division of the spermatocytes and passes undivided to one cell in the second division. In this it moves with its longer axis parallelly to the spindle axis and shows fibrillar connections with both poles. Special attention was paid to the behavior of the chromosomes in the anaphase of the spermatogonia. As it was previously stated (Piza 1946 and 1946a) with regard to other species, the chromosomes are here attached to the spindle by both ends and begin to move toward the poles strongly curved to them. No intercalary fibers could be detected although their existente may not be denied by theoretical reasons developed in another paper (Piza 1946). Mitoses in somatic tissues of the embryo were equally studied. Careful examination of anaphase chromosomes in a great number of cells showed that the chromosomes behave exactly as in the spermatogonia, being equally attached to the spindle by the extremities alone and moving with their ends looking to the pole. A weak median constriction sometimes replaced by a slightly clearer space was observed in prometaphase and even in metaphase chromosomes of the spermatogonia as well as the somatic cells, having already been referred to in the case of Diactor bilineatus. (Piza 1945). Hemipteran chromosomes being considered as iso-chromosomes originated by a longitudinal spliting of the monocentric chromosomes resulting from the second division of the spermatocytes, the median aspect just mentioned may be regarded as the point of union of the separated halves. (See origin of dicentricity in Piza 1946). b) Leptoglossus stigma - This species has spermatogonia provided with 20 pairs of autosomes and one sex chromosome whose behavior differs in nothing from what was stated in regard of the preceding species. In the primary spermatocytes nothing meriting special mention was observed. Orientation, connection with the poles and movements of the sex chromosome in the secondary spermatocytes confirm the views already developed.

Nota prévia sôbre a meiose de Corizus (Liorhyssus) hyalinus (Fabr.) (Hemiptera-Corizidae)

The main facts presented in this paper may be summarized as follows: 1) Corizus (Liorhyssus) hyalinus (Fabr.) has primary spermatocytes provided with 6 autosomal tetrads, one pair of microchromosomes and one sex chromosome. 2) The two microchromosomes present in this species sometimes appear at the primary metaphase as an unequal pair of minute elements. In the secondary spermatocytes the unique microchromosome present may be in the limit of visibility or entirely invisible. This invisibility may be partly due to a loss of colourability. 3) The sex chromosome divides transversely in the first division of the spermatocyte, passing undivided to one pole in the second one. In the latter it becomes fusiform in the beginning of anaphase revealing in this manner its dicentricity. In late anaphase it finishes by passing to one pole leaving in the other pole one of its kinetochores sometimes accompanied by a chromosomal fragment. 4) All the chromosomes divide transversely in both divisions, a diagram being enclosed to elucidate the question. 5) Spermatogonial chromosomes are provided with one kinetochore at each end, being curved toward the poles since the most beginning anaphase. 6) The following hypothesis is presented as an essay to explain the origin of microchromosomes: Since microchromosomes parallel sex chromosomes in most respects, as for instances in heteropycnosis and pairing modus, it seems highly probable that they originate from sex chromosomes. One may suppose that the ancestral form of a given species had a sex chromosome which used to lose a small centric fragment when it divided during meiosis. This fragment might well be at first an unstable one. Later, to compensate the effects of such a deficiency a mechanism arose through evolution which produced two useful results : a) the establishment of the fragment as a permanent structure of the cell nucleus and b) the acquirement by the sex chromosome of the faculty of passing to one pole without losing any of its ends.

Notas sobre os cromossomios dos proscopiidios

Cephalocoema borellii (Giglio-Tos) has 19 chromosomes, that is 9 pairs of autosomes and a single heterochromosome, the latter having been observed either in succession or in synchronism as was the case of the two other species studied by Prof. PIZA, namely Cephalocoema zilkari Piza and Tetanorhynchus mendesi Piza. (= Cephalocoema sica Serv.).

Soldadura por uma das extremidades de dois cromossômios homólogos do tityus

In this paper the author describes a very interesting case of union of two homologous chromosomes of the scorpion Tityus bahiensis just by the opposite extremities. The two normal pairs of chromosomes behave as ordinarily, the members of each pair showing at times a slight disturbance in their regular parallelism. The complex chromosome, on the contrary, behaves itself as if it were devoid of kinetochores, that is, it does not orient like normal chromosomes nor reveal any kind of active movement. The fusion of the chromosomes has resulted from terminal breakage at the opposite ends, the correspondig fragments having been found unpaired in a cell in which two pairs of chromosomes were present. Consequently, the compound chromosome, like the normal ones, is provided with a kinetochore at each one of the free ends. Being thus a centric chromosome its behavior, or more exactly, its kinetic inactivity may be compared with that of the monovalents found elsewhere in meioses. It is due o the failure of a partner. The fusion of two homologous chromosomes has transformed them into a new chromosomal unit in whose corresponding parts the ability of pairing was entirely abolished. This result is in full contradiction with the theory of a point-to point attraction between homologous chromosomes attributed to particular power of the genes, since, if genes really exist, being placed in their original loci, they would promote the union side by side of the members of the compound chromosome. If an attraction loci-to-loci should prevail the compound chromosome would be bent as in Fig. 8, C or form a ring similar to the loops observed in the inverted segment of sailvary chromosomes of Drosophila, as represented in the Fig. 8, D and this, in accordance with the order of the loci resulting from an union of corresponding or opposite ends of the fused chromosomes, as indicated in the Fig, 8 A and B. The evidence in hand points to a fusion by non homologous extremities. The expected rings, however, have never been found in metaphase plates. From this fact the author concludes that there is no point-to-point attraction between chromosomes, a conclusion in full agreement with the behavior of Hemipteran chromosomes which, in spite of geing composed of two equivalent halves do not bend in order to adjust the corresponding loci. (Cf. the papers on Hemiptera published by the author in this volume).

Aspectos interessantes observados na meiose de alguns hemípteros

Particular aspects of the meiosis of two species of Hemiptera, namely Megalotomus pallescens (Stal) (Coriscidae) and Jadera sanguinolenta (Fabr.); (Corizidae) are described and discussed in this paper. Megalotomus pallescens This species has primary spermatocytes provided with 7 autosomal tetrads plus a single sex chromosome. The X is smaller than the autosomes and may be found either in the periphery of the circle formed by the autosomal tetrads or in the center together with the m-tetrad which always occupies this position. The X chromosome - In the primary spermatocytes this element, which is tetradiform, orients itself parallelly to the spindle axis and divides transversely by its median constriction. In the secondary spermatocytes it passes undivided to one pole. The m-chromosomes - These chromosomes have been frequently found in close association with the sex chromosome in nuclei wich have passed the diffuse stage, a fact which was considered as affording some evidence in support of the idea /developed by the present writer in another paper with regard to the origin of the m-chromosomes from the sex chromosome. Formation of tetrads - Tetrads appear at first as irregular areas of reticular structure, becoming later more and more distinct. Then, two chromosomal strands very loose and irregular in outline, connected whit each other by several transverse filaments, begin to develop in each area. Growing progressively shorter, thicker and denser, these strands soon give origin to typical Hemiptera tetrads. Jadera sanguinolenta Spermatogonia of this species have 13 chromosomes, that is, 10 autosomes, 2 m-chromosomes and one sex chromosome, one pair of autosomes being much larger than the rest. Chromosomes move toward the poles with both ends looking to them. Primary spermatocytes show 6 tetrads and a single X. The sex chromossome in the first division of the spermatocytes divides as if it was a tetrad, passing undivided to one pole in the second division. In the latter it does not orient, being found anywhere in the cells. Its most common situation in anaphase corresponds therefore to precession. Tetrads are formed here in an entirely different way : the bivalents as they become distinct in the nuclei which came out. of the diffuse stage they appear in form of two thin threads united only at the extremities, an aspect which may better be analized in the larger bivalent. Up from this stage the formation of the tetrads is a mere process of shortening and thickening of both members of the pair. Due to the fact that the paired chromosomes are well separated from each other throughout their entire lenght, the author concluded that chiasmata, if present, are accumulated at the very ends of the bivalents. If no chiasmata have been at all formed, then, what holds together the corresponding extremities must be a strong attraction developed by the kinetochores. If one interprets the bivalents represented in the figures 17-21 as formed by four chromatids paired by one of the ends and united by the opposite one, then the question of the diffuse attachment becomes entirely disproved since it is exactly by the distal extremities that the tetrads later will be connected with the poles. In the opinion of the present writer the facts referred to above are one of the best demonstration at hand of the continuity of the paired threads and at the same time of the dicentricity of Hemiptera chromosomes. In view of the data hitherto collected by the author the behavior of the sex chromosome of the Hemiptera whose males are of the XO type may be summarized as follows: a) The sex chromosome in the primary metaphase appears longitudinally divided, without transverse constriction. It is oriented with the extremities in the plane of the equator and its chromatids separate by the plane of division. (Euryophthalmus, Protenor). In the second division the sex chromosome, provided as it is with an active kinetochore at each end, orients itself with its lenght parallelly to the spindle axis and passes undivided to one pole (Protenor?), or loses to the other pole a centric end (Euryophthalmus) In the latter case it has to become dicentric by means of a longitudinal spliting beginning at the kinetochore. b) The sex chromosome in the primary metaphase is tetradiform, that is, it is provided with a longitudinal split and a median transverse constriction. Orients with its length paral lelly to the spindle axis (what is probably due to the kinetochores being not yet divided) and divides transversely. (Corizas hyalinus, Megalotomus pallescens). in the secondary metaphase the sex chromosome which turned to be dicentric in consequence of a longitudinal spliting initiated in the kineto chore, orients perpendicularly to the equatorial plane and without losing anyone of its extremities passes undivided to one pole (Megalotomus). Or, distending between both poles passes to one side, in which case it loses one of its ends to the other side. (Corizas hyalinus). c) The very short sex chromosome in the first division of the spermatocytes orients in the same manner aa the tetrads and divides transversely. In the second division, due to the inactivity o the inetochore, it remains monocentric and motionless anywhere in the cell, finishing by being enclosed in the nearer nucleus. In the secondary telophase it recuperates its dicentricity at the same time as the autosomal chromatids. (Jadera sanguinolenta, Diactor bilineatus). d) The sex chromosome in the first division orients in the equador with its longitudinal axis parallelly to the spindle axis passing integrally to one pole or, distending itself between the anaphase plates, loses one of its ends to the opposite pole. In this case it becomes dicentric in the prometaphase of the second division, behaving in this division as the autossomes. It thus divides longitudnally. (Pachylis laticomis, Pachylis pharaonis).