Study of antiretroviral mutants in HIV patients with treatment failures and the effect of risk factors in the virological failures

INTRODUCTION: Information about HIV phenotypes of resistant to available ART and the influence of different risk factors on virological failures (VF) in Costa Rican HIV positive patients prior or during HAART is unknown. MATERIALS AND METHODS: Eighty nine samples, 72 VF and 17 basal (before treatment) were analyzed by examining resistant mutants in reverse transcriptase (RT) and protease (PT) regions using Trugene or LIPA genotyping kits. Sixty eight control patients were selected and relevant information was collected in a questionnaire. RESULTS: Poor adherence, presence of resistant mutations and number of treatment's changes were the only significant factors found (p = 0.006, 0.04 and 0.01 respectively). From 66 sequenced samples, 78%, 50% and 50% showed resistance to NRTI (nucleoside reverse transcriptase inhibitors), NNRT (non-nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors), respectively. The most frequent mutations were M41L, M184V, and T215FY in RT and L62PI, L10FIRV and M36I in PT. DISCUSSION: The most important factor related to treatment response in this study was adherence to treatment. Mutations in RT were related to the treatment failure while the ones found in PT were secondary mutations which have been previously described to influence the selection of primary resistance mutations in these regions. The study reveals the urgency to detect resistant mutations in VF to be considered by physicians for selection of treatment schedule, to analyze basal HIV patients for monitoring of the spread of resistant mutations and the importance to reinforce the adherence in the patients for overall treatment outcome.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

The emergence of YMDD mutants precedes biochemical flare by 19 weeks in lamivudine-treated chronic hepatitis B patients: an opportunity for therapy reevaluation

Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 ± 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 ± 14 and 60 ± 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 ± 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

GB virus C/Hepatitis G virus and other putative hepatitis non A-E viruses

The identification of the major agents causing human hepatitis (Hepatitis A, B, C, D and E Viruses) was achieved during the last 30 years. These viruses are responsible for the vast majority of human viral hepatitis cases, but there are still some cases epidemiologically related to infectious agents without any evidence of infection with known virus, designated as hepatitis non A - E. Those cases are considered to be associated with at least three different viruses: 1 - Hepatitis B Virus mutants expressing its surface antigen (HBsAg) with altered epitopes or in low quantities; 2 - Another virus probably associated with enteral transmitted non A-E hepatitis, called Hepatitis F Virus. Still more studies are necessary to better characterize this agent; 3 - Hepatitis G Virus or GB virus C, recently identified throughout the world (including Brazil) as a Flavivirus responsible for about 10% of parenteral transmitted hepatitis non A-E. Probably still other unknown viruses are responsible for human hepatitis cases without evidence of infection by any of these viruses, that could be called as non A-G hepatitis.

Hepatitis C viral load does not predict disease outcome: going beyond numbers

The analysis of 58 patients with chronic hepatitis C without cirrhosis and treated with interferon-alpha demonstrated that hepatitis C viral (HCV) load does not correlate with the histological evolution of the disease (p = 0.6559 for architectural alterations and p = 0.6271 for the histological activity index). Therefore, the use of viral RNA quantification as an evolutive predictor or determinant of the severity of hepatitis C is incorrect and of relative value. A review of the literature provided fundamental and interdependent HCV (genotype, heterogeneity and mutants, specific proteins), host (sex, age, weight, etc) and treatment variables (dosage, time of treatment, type of interferon) within the broader context of viral kinetics, interferon-mediated immunological response (in addition to natural immunity against HCV) and the role of interferon as a modulator of fibrogenesis. Therefore, viral load implies much more than numbers and the correct interpretation of these data should consider a broader context depending on multiple factors that are more complex than the simple value obtained upon quantification.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

Enterobacteriaceae ISOLATES FROM THE ORAL CAVITY OF WORKERS IN A BRAZILIAN ONCOLOGY HOSPITAL

The evaluation of workers as potential reservoirs and disseminators of pathogenic bacteria has been described as a strategy for the prevention and control of healthcare-associated infections (HAIs). The aim of this study was to evaluate the presence of Enterobacteriaceae in the oral cavity of workers at an oncology hospital in the Midwest region of Brazil, as well as to characterize the phenotypic profile of the isolates. Saliva samples of 294 workers from the hospital’s healthcare and support teams were collected. Microbiological procedures were performed according to standard techniques. Among the participants, 55 (18.7%) were colonized by Enterobacteriaceae in the oral cavity. A total of 64 bacteria were isolated, including potentially pathogenic species. The most prevalent species was Enterobacter gergoviae (17.2%). The highest rates of resistance were observed for β-lactams, and 48.4% of the isolates were considered multiresistant. Regarding the enterobacteria isolated, the production of ESBL and KPC was negative. Nevertheless, among the 43 isolates of the CESP group, 51.2% were considered AmpC β-lactamase producers by induction, and 48.8% were hyper-producing mutants. The significant prevalence of carriers of Enterobacteriaceae and the phenotypic profile of the isolates represents a concern, especially due to the multiresistance and production of AmpC β-lactamases.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. Conclusions HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

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 experimentais sôbre a origem do milho

1) It may seem rather strange that, in spite of the efforts of a considerable number of scientists, the problem of the origin of indian corn or maize still has remained an open question. There are no fossil remains or archaeological relics except those which are quite identical with types still existing. (Fig. 1). The main difficulty in finding the wild ancestor- which may still exist - results from the fact that it has been somewhat difficult to decide what it should be like and also where to look for it. 2) There is no need to discuss the literature since an excellent review has recently been published by MANGELSDORF and REEVES (1939). It may be sufficient to state that there are basically two hypotheses, that of ST. HILAIRE (1829) who considered Brazilian pod corn as the nearest relative of wild corn still existing, and that of ASCHERSON (1875) who considered Euchlaena from Central America as the wild ancestor of corn. Later hypotheses represent or variants of these two hypotheses or of other concepts, howewer generally with neither disproving their predecessors nor showing why the new hypotheses were better than the older ones. Since nearly all possible combinations of ideas have thus been put forward, it har- dly seems possible to find something theoretically new, while it is essential first to produce new facts. 3) The studies about the origin of maize received a new impulse from MANGELSDORF and REEVES'S experimental work on both Zea-Tripsacum and Zea-Euchlaena hybrids. Independently I started experiments in 1937 with the hope that new results might be obtained when using South American material. Having lost priority in some respects I decided to withold publication untill now, when I can put forward more concise ideas about the origin of maize, based on a new experimental reconstruction of the "wild type". 4) The two main aspects of MANGELSDORF and REEVES hypothesis are discussed. We agree with the authors that ST. HILAIRE's theory is probably correct in so far as the tunicata gene is a wild type relic gene, but cannot accept the reconstruction of wild corn as a homozygous pod corn with a hermaphroditic tassel. As shown experimentally (Fig. 2-3) these tassels have their central spike transformed into a terminal, many rowed ear with a flexible rachis, while possessing at the same time the lateral ear. Thus no explanation is given of the origin of the corn ear, which is the main feature of cultivated corn (BRIEGER, 1943). The second part of the hypothesis referring to the origin of Euchlaena from corn, inverting thus ASCHERSON's theory, cannot be accepted for several reasons, stated in some detail. The data at hand justify only the conclusion that both genera, Euchlaena and Zea, are related, and there is as little proof for considering the former as ancestor of the latter as there is for the new inverse theory. 5) The analysis of indigenous corn, which will be published in detail by BRIEGER and CUTLER, showed several very primitive characters, but no type was found which was in all characters sufficiently primitive. A genetical analysis of Paulista Pod Corn showed that it contains the same gene as other tunicates, in the IV chromosome, the segregation being complicated by a new gametophyte factor Ga3. The full results of this analysis shall be published elsewhere. (BRIEGER). Selection experiments with Paulista Pod Corn showed that no approximation to a wild ancestor may be obtained when limiting the studies to pure corn. Thus it seemed necessary to substitute "domesticated" by "wild type" modifiers, and the only means for achieving this substitution are hybridizations with Euchlaena. These hybrids have now been analysed init fourth generation, including backcrosses, and, again, the full data will be published elsewhere, by BRIEGER and ADDISON. In one present publication three forms obtained will be described only, which represent an approximation to wild type corn. 6) Before entering howewer into detail, some arguments against ST. HILAIRE's theory must be mentioned. The premendelian argument, referring to the instability of this character, is explained by the fact that all fertile pod corn plants are heterozygous for the dominant Tu factor. But the sterility of the homozygous TuTu, which phenotypically cannot be identified, is still unexplained. The most important argument against the acceptance of the Tunicata faetor as wild type relic gene was removed recently by CUTLER (not yet published) who showed that this type has been preserved for centuries by the Bolivian indians as a mystical "medicine". 7) The main botanical requirements for transforming the corn ear into a wild type structure are stated, and alternative solutions given. One series of these characters are found in Tripsacum and Euchlaena : 2 rows on opposite sides of the rachis, protection of the grains by scales, fragility of the rachis. There remains the other alternative : 4 rows, possibly forming double rows of female and male spikelets, protection of kernels by their glumes, separation of grains at their base from the cob which is thin and flexible. 8) Three successive stages in the reconstruction of wild corn, obtained experimentally, are discussed and illustrated, all characterized by the presence of the Tu gene. a) The structure of the Fl hybrids has already been described in 1943. The main features of the Tunicata hybrids (Fig. -8), when compared with non-tunicate hybrids (Fig. 5-6), consist in the absence of scaly protections, the fragility of the rachis and finally the differentiation of the double rows into one male and one female spikelet. As has been pointed out, these characters represent new phenotypic effects of the tunicate factor which do not appear in the presence of pure maize modifiers. b) The next step was observed among the first backcross to teosinte (Fig. 9). As shown in the photography, Fig. 9D, the features are essencially those of the Fl plants, except that the rachis is more teosinte like, with longer internodes, irregular four-row-arrangement and a complete fragility on the nodes. c) In the next generation a completely new type appeared (Fig. 10) which resembles neither corn nor teosinte, mainly in consequence of one character: the rachis is thin and flexible and not fragile, while the grains have an abscission layer at the base, The medium sized, pointed, brownish and hard granis are protected by their well developed corneous glumes. This last form may not yet be the nearest approach to a wild grass, and I shall try in further experiments to introduce other changes such as an increase of fertile flowers per spikelet, the reduction of difference between terminal and lateral inflorescences, etc.. But the nature of the atavistic reversion is alveadwy such that it alters considerably our expectation when looking for a still existing wild ancestor of corn. 9) The next step in our deductions must now consist in an reversion of our question. We must now explain how we may obtain domesticated corn, starting from a hypothetical wild plant, similar to type c. Of the several changes which must have been necessary to attract the attention of the Indians, the following two seem to me the most important: the disappearance of all abscission layers and the reduction of the glumes. This may have been brought about by an accumulation of mutations. But it seems much more probable to assume that some crossing with a tripsacoid grass or even with Tripsacum australe may have been responsible. In such a cross, the two types of abscission layer would be counterbalanced as shown by the Flhybrids of corn, Tripsacum and Euchlaena. Furthermore in later generations a.tu-allele of Tripsacum may become homozygous and substitute the wild tunicate factor of corn. The hypothesis of a hybrid origin of cultivated corn is not completely new, but has been discussed already by HARSHBERGER and COLLINS. Our hypothesis differs from that of MANGELSDORF and REEVES who assume that crosses with Tripsacum are responsible only for some features of Central and North American corn. 10) The following arguments give indirects evidence in support of our hypothesis: a) Several characters have been observed in indigenous corn from the central region of South America, which may be interpreted as "tripsacoid". b) Equally "zeoid" characters seem to be present in Tripsacum australe of central South-America. c) A system of unbalanced factors, combined by the in-tergeneric cross, may be responsible for the sterility of the wild type tunicata factor when homozygous, a result of the action of modifiers, brought in from Tripsacum together with the tuallele. d) The hybrid theory may explain satisfactorily the presence of so many lethals and semilethals, responsible for the phenomenon of inbreeding in cultivated corn. It must be emphasized that corn does not possess any efficient mechanism to prevent crossing and which could explain the accumulation of these mutants during the evolutionary process. Teosinte which'has about the same mechanism of sexual reproduction has not accumulated such genes, nor self-sterile plants in spite of their pronounced preference for crossing. 11) The second most important step in domestication must have consisted in transforming a four rowed ear into an ear with many rows. The fusion theory, recently revived byLANGHAM is rejected. What happened evidently, just as in succulent pXants (Cactus) or in cones os Gymnosperms, is that there has been a change in phyllotaxy and a symmetry of longitudinal rows superimposed on the original spiral arrangement. 12) The geographical distribution of indigenous corn in South America has been discussed. So far, we may distinguish three zones. The most primitive corn appears in the central lowlands of what I call the Central Triangle of South America: east of the Andies, south of the Amazone-Basin, Northwest of a line formed by the rivers São Prancisco-Paraná and including the Paraguay-Basin. The uniformity of the types found in this extremely large zone is astonishing (BRIEGER and CUTLER). To the west, there is the well known Andian region, characterized by a large number of extremely diverse types from small pop corn to large Cuszco, from soft starch to modified sweet corn, from large cylindrical ears to small round ears, etc.. The third region extends along the atlantic coast in the east, from the Caribean Sea to the Argentine, and is characterized by Cateto, an orange hard flint corn. The Andean types must have been obtained very early, and undoubtedly are the result of the intense Inca agriculture. The Cateto type may be obtained easily by crosses, for instance, of "São Paulo Pointed Pop" to some orange soft corn of the central region. The relation of these three South American zones to Central and North America are not discussed, and it seems essential first to study the intermediate region of Ecuador, Colombia and Venezuela. The geograprical distribution of chromosome knobs is rapidly discussed; but it seems that no conclusions can be drawn before a large number of Tripsacum species has been analysed.

Bases para o estudo da genética de populações dos Hymenoptera em geral e dos Apinae sociais em particular

This paper deals with problems on population genetics in Hymenoptera and particularly in social Apidae. 1) The studies on populations of Hymenoptera were made according to the two basic types of reproduction: endogamy and panmixia. The populations of social Apinae have a mixed method of reproduction with higher percentage of panmixia and a lower of endogamy. This is shown by the following a) males can enter any hive in swarming time; b) males of Meliponini are expelled from hives which does not need them, and thus, are forced to look for some other place; c) Meliponini males were seen powdering themselves with pollen, thus becoming more acceptable in any other hive. The panmixia is not complete owing to the fact that the density of the breeding population as very low, even in the more frequent species as low as about 2 females and 160 males per reproductive area. We adopted as selection values (or survival indices) the expressions according to Brieger (1948,1950) which may be summarised as follows; a population: p2AA + ²pq Aa + q2aa became after selection: x p2AA + 2pq Aa + z q²aa. For alge-braics facilities Brieger divided the three selective values by y giving thus: x/y p2 AA + y/y 2 pq Aa + z/y q²aa. He called x/y of RA and z/y of Ra, that are survival or selective index, calculated in relation to the heterozygote. In our case all index were calculated in relation to the heterozygote, including the ones for haploid males; thus we have: RA surveval index of genotype AA Ra surveval index of genotype aa R'A surveval index of genotype A R'a surveval index of genotype a 1 surveval index of genotype Aa The index R'A ande R'a were equalized to RA and Ra, respectively, for facilities in the conclusions. 2) Panmitic populations of Hymenoptera, barring mutations, migrations and selection, should follow the Hardy-Weinberg law, thus all gens will be present in the population in the inicial frequency (see Graphifc 1). 3) Heterotic genes: If mutation for heterotic gene ( 1 > RA > Ra) occurs, an equilibrium will be reached in a population when: P = R A + Ra - 2R²a _____________ (9) 2(R A + Ra - R²A - R²a q = R A + Ra - 2R²A _____________ (10) 2(R A + Ra - R²A - R²a A heterotic gene in an hymenopteran population may be maintained without the aid of new mutation only if the survival index of the most viable mutant (RA) does not exced the limiting value given by the formula: R A = 1 + √1+Ra _________ 4 If RA has a value higher thah the one permitted by the formula, then only the more viable gene will remain present in the population (see Graphic 10). The only direct proof for heterotic genes in Hymenoptera was given by Mackensen and Roberts, who obtained offspring from Apis mellefera L. queens fertilized by their own sons. Such inbreeding resulted in a rapid loss of vigor the colony; inbred lines intercrossed gave a high hybrid vigor. Other fats correlated with the "heterosis" problem are; a) In a colony M. quadrifasciata Lep., which suffered severely from heat, the percentage of deths omong males was greater .than among females; b) Casteel and Phillips had shown that in their samples (Apis melifera L). the males had 7 times more abnormalities tian the workers (see Quadros IV to VIII); c) just after emerging the males have great variation, but the older ones show a variation equal to that of workers; d) The tongue lenght of males of Apis mellifera L., of Bombus rubicundus Smith (Quadro X), of Melipona marginata Lep. (Quadro XI), and of Melipona quadrifasciata Lep. Quadro IX, show greater variationthan that of workers of the respective species. If such variation were only caused by subviables genes a rapid increasse of homozigoty for the most viable alleles should be expected; then, these .wild populations, supposed to be in equilibrium, could .not show such variability among males. Thus we conclude that heterotic genes have a grat importance in these cases. 4) By means of mathematical models, we came to the conclusion tht isolating genes (Ra ^ Ra > 1), even in the case of mutations with more adaptability, have only the opor-tunity of survival when the population number is very low (thus the frequency of the gene in the breeding population will be large just after its appearence). A pair of such alleles can only remain present in a population when in border regions of two races or subspecies. For more details see Graphics 5 to 8. 5) Sex-limited genes affecting only females, are of great importance toHymenoptera, being subject to the same limits and formulas as diploid panmitic populations (see formulas 12 and 13). The following examples of these genes were given: a) caste-determining genes in the genus Melipona; b) genes permiting an easy response of females to differences in feeding in almost all social Hymenoptera; c) two genes, found in wild populations, one in Trigona (Plebéia) mosquito F. SMITH (quadro XII) and other in Melipona marginata marginata LEP. (Quadro XIII, colonies 76 and 56) showing sex-limited effects. Sex-limited genes affecting only males do not contribute to the plasticity or genie reserve in hymenopteran populations (see formula 14). 6) The factor time (life span) in Hymenoptera has a particular importance for heterotic genes. Supposing one year to be the time unit and a pair of heterotic genes with respective survival indice equal to RA = 0, 90 and Ra = 0,70 to be present; then if the life time of a population is either one or two years, only the more viable gene will remain present (see formula 11). If the species has a life time of three years, then both alleles will be maintained. Thus we conclude that in specis with long lif-time, the heterotic genes have more importance, and should be found more easily. 7) The colonies of social Hymenoptera behave as units in competition, thus in the studies of populations one must determine the survival index, of these units which may be subdivided in indice for egg-laying, for adaptive value of the queen, for working capacity of workers, etc. 8) A study of endogamic hymenopteran populations, reproduced by sister x brother mating (fig. 2), lead us to the following conclusions: a) without selection, a population, heterozygous for one pair of alleles, will consist after some generations (theoretically after an infinite number of generation) of females AA fecundated with males A and females aa fecundated with males a (see Quadro I). b) Even in endogamic population there is the theoretical possibility of the presence of heterotic genes, at equilibrium without the aid of new mutations (see Graphics 11 and 12), but the following! conditions must be satisfied: I - surveval index of both homozygotes (RA e Ra) should be below 0,75 (see Graphic 13); II - The most viable allele must riot exced the less viable one by more than is permited by the following formula (Pimentel Gomes 1950) (see Gra-fic 14) : 4 R5A + 8 Ra R4A - 4 Ra R³A (Ra - 1) R²A - - R²a (4 R²a + 4 Ra - 1) R A + 2 R³a < o Considering these two conditions, the existance of heterotic genes in endogamic populations of Hymenoptera \>ecames very improbable though not - impossible. 9) Genie mutation offects more hymenopteran than diploid populations. Thus we have for lethal genes in diploid populations: u = q2, and in Hymenoptera: u = s, being u the mutation ratio and s the frequency of the mutant in the male population. 10) Three factors, important to competition among species of Meliponini were analysed: flying capacity of workers, food gathering capacity of workers, egg-laying of the queen. In this connection we refer to the variability of the tongue lenght observed in colonies from several localites, to the method of transporting the pollen in the stomach, from some pots (Melliponi-ni storage alveolus) to others (e. g. in cases of pillage), and to the observation that the species with the most populous hives are almost always the most frequent ones also. 11) Several defensive ways used for Meliponini to avoid predation are cited, but special references are made upon the camouflage of both hive (fig. 5) and hive entrance (fig. 4) and on the mimetism (see list in page ). Also under the same heading we described the method of Lestrimelitta for pillage. 12) As mechanisms important for promoting genetic plasticity of hymenopteran species we cited: a) cytological variations and b) genie reserve. As to the former, duplications and numerical variations of chromosomes were studied. Diprion simile ATC was cited as example for polyploidy. Apis mellife-ra L. (n •= 16) also sugests polyploid origen since: a) The genus Melipona, which belongs to a" related tribe, presents in all species so far studied n = 9 chromosomes and b) there occurs formation of dyads in the firt spermatocyte division. It is su-gested that the origin of the sex-chromosome of Apis mellifera It. may be related to the possible origin of diplo-tetraploidy in this species. With regards to the genie reserve, several possible types of mutants were discussed. They were classified according to their survival indices; the heterotic and neutral mutants must be considered as more important for the genie reserve. 13) The mean radius from a mother to a daghter colony was estimated as 100 meters. Since the Meliponini hives swarm only once a year we may take 100 meters a year as the average dispersion of female Meliponini in ocordance to data obtained from Trigona (tetragonisca) jaty F. SMITH and Melipona marginata LEP., while other species may give different values. For males the flying distance was roughly estimated to be 10 times that for females. A review of the bibliography on Meliponini swarm was made (pg. 43 to 47) and new facts added. The population desity (breeding population) corresponds in may species of Meliponini to one male and one female per 10.000 square meters. Apparently the males are more frequent than the females, because there are sometimes many thousands, of males in a swarm; but for the genie frequency the individuals which have descendants are the ones computed. In the case of Apini and Meliponini, only one queen per hive and the males represented by. the spermatozoos in its spermateca are computed. In Meliponini only one male mate with the queen, while queens of Apis mellijera L. are fecundated by an average of about 1, 5 males. (Roberts, 1944). From the date cited, one clearly sees that, on the whole, populations of wild social bees (Meliponini) are so small that the Sewall Wright effect may become of great importance. In fact applying the Wright's formula: f = ( 1/aN♂ + 1/aN♀) (1 - 1/aN♂ + 1/aN♀) which measures the fixation and loss of genes per generation, we see that the fixation or loss of genes is of about 7% in the more frequent species, and rarer species about 11%. The variation in size, tergite color, background color, etc, of Melipona marginata Lep. is atributed to this genetic drift. A detail, important to the survival of Meliponini species, is the Constance of their breeding population. This Constance is due to the social organization, i. e., to the care given to the reproductive individuals (the queen with its sperm pack), to the way of swarming, to the food storage intended to control variations of feeding supply, etc. 14) Some species of the Meliponini are adapted to various ecological conditions and inhabit large geographical areas (e. g. T. (Tetragonisca jaty F. SMITH), and Trigona (Nanno-trigona testaceicornis LEP.) while others are limited to narrow regions with special ecological conditions (e. g. M. fuscata me-lanoventer SCHWARZ). Other species still, within the same geographical region, profit different ecological conditions, as do M. marginata LEP. and M. quadrifasciata LEP. The geographical distribution of Melipona quadrifasciata LEP. is different according to the subspecies: a) subsp anthidio-des LEP. (represented in Fig. 7 by black squares) inhabits a region fron the North of the S. Paulo State to Northeastern Brazil, ,b) subspecies quadrifasciata LEP., (marked in Fig. 7 with black triangles) accurs from the South of S. Paulo State to the middle of the State of Rio Grande do Sul (South Brazil). In the margined region between these two areas of distribution, hi-brid colonies were found (Fig. 7, white circles); they are shown with more details in fig. 8, while the zone of hybridization is roughly indicated in fig. 9 (gray zone). The subspecies quadrifasciata LEP., has 4 complete yellow bands on the abdominal tergites while anthidioides LEP. has interrupted ones. This character is determined by one or two genes and gives different adaptative properties to the subspecies. Figs. 10 shows certains meteorological isoclines which have aproximately the same configuration as the limits of the hybrid zone, suggesting different climatic adaptabilities for both genotypes. The exis-tance of a border zone between the areas of both subspecies, where were found a high frequency of hybrids, is explained as follows: being each subspecies adapted to a special climatic zone, we may suppose a poor adaptation of either one in the border region, which is also a region of intermediate climatic conditions. Thus, the hybrids, having a combination of the parent qualities, will be best adapted to the transition zone. Thus, the hybrids will become heterotic and an equilibrium will be reached with all genotypes present in the population in the border region.