DNA sequence analysis and the phylogeographical history of the rodent Deltamys kempi (Sigmodontinae, Cricetidae) on the Atlantic Coastal Plain of south of Brazil.

The rodent Deltamys kempiThomas, 1917 is found on the Coastal Plain - a recently formed geographic region located on Brazil's south-east coast. Considering that Deltamys is the only South American sigmodontine with a sex chromosome system of type X(1)X(1)X(2)X(2)/X(1)X(2)Y, this investigation was focused on the phylogeographic history of this taxon by using gene sequence analysis, trying to clarify when Deltamys differentiated, what was its centre of diversification, and what were the probable routes it used to reach its present distribution. We analysed sequences of the mitochondrial cytochrome b gene and nuclear recombination activating gene 2, performed cranial measurements and searched for centric fusions in individuals collected in distinct localities. The results, clearly demonstrate that D. kempi, on the Coastal Plain, divided into two groups, one occupying a small portion to the north of this region and the other spreading widely to the south. In this process, the phenomena of marine transgression and regressions which moulded its habitat, together with the occurrence of successive chromosomal rearrangements, were certainly the fundamental factors in shaping D. kempi diversification.

Genetic diversity and relatedness within and between species of the genus Oligoryzomys (Rodentia; Sigmodontinae).

A RAPD analysis on six species of the rodent genus Oligoryzomys trapped in a wide area (ranging from 01 degrees N to 32 degrees S) of Brazilian territory was performed in order to determine the levels of genetic variability within and between its populations and species. One-hundred and ninety-three animals were collected in 13 different sites (corresponding to 17 samples) located at Pampas, Atlantic Rain Forest, Cerrado, and Amazon domains. Oligoryzomys sp., O. nigripes (8 populations), O. flavescens (4 populations), O. moojeni, O. stramineus, and O. fornesi were the taxa analyzed. Of the 20 primers tested, 4 generated a total of 75 polymorphic products simultaneously amplified in 151 specimens. Various diversity estimators analyzed showed considerable differences between species and populations, indicating a great genetic variation occurring in the Oligoryzomys taxa investigated. A cluster analysis was made using Nei's standard genetic distances, however, it did not correlate the genetic heterogeneity of the species and populations with the geographical areas.

Genetic diversity and relatedness within and between species of the genus Oligoryzomys (Rodentia; Sigmodontinae)

A RAPD analysis on six species of the rodent genus Oligoryzomys trapped in a wide area (ranging from 01° N to 32° S) of Brazilian territory was performed in order to determine the levels of genetic variability within and between its populations and species. One-hundred and ninety-three animals were collected in 13 different sites (corresponding to 17 samples) located at Pampas, Atlantic Rain Forest, Cerrado, and Amazon domains. Oligoryzomys sp., O. nigripes (8 populations), O. flavescens (4 populations), O. moojeni, O. stramineus, and O. fornesi were the taxa analyzed. Of the 20 primers tested, 4 generated a total of 75 polymorphic products simultaneously amplified in 151 specimens. Various diversity estimators analyzed showed considerable differences between species and populations, indicating a great genetic variation occurring in the Oligoryzomys taxa investigated. A cluster analysis was made using Nei's standard genetic distances, however, it did not correlate the genetic heterogeneity of the species and populations with the geographical areas.

Foram realizadas análises com RAPD em seis espécies de roedores do gênero Oligoryzomys capturados em uma ampla área (estendendo-se de 01° N a 32° S) do território brasileiro com o objetivo de determinar os níveis de variabilidade genética dentro e entre as populações e espécies. Cento e noventa e três animais foram coletados em 13 locais diferentes (correspondendo a 17 amostras) localizados nos Pampas, Floresta Atlântica, Cerrado e Amazônia. Oligoryzomys sp., O. nigripes (8 populações), O. flavescens (4 populações), O. moojeni, O. stramineus e O. fornesi foram as espécies analisadas. Vinte primers foram testados, sendo que quatro deles geraram um total de 75 produtos polimórficos amplificados simultaneamente em 151 exemplares. Várias estimativas de diversidade apresentaram diferenças consideráveis entre as espécies e as populações, indicando uma grande variação genética entre os taxa de Oligoryzomys investigados. As análises de agrupamento utilizando a distância genética de Nei, entretanto, não correlacionaram a heterogeneidade genética das espécies e populações com as áreas geográficas.

Prevalence of TEL/AML1 fusion gene in Brazilian pediatric patients with acute lymphoblastic leukemia.

We studied 58 childhood B-lineage acute lymphoblastic leukemia (B-ALL) in Brazilian sample patients at the time of diagnosis to investigate the prevalence of the cryptic t(12;21)(p13;q22). All bone marrow specimens were G-band karyotyped, and commercial dual-color DNA probes were used to search for fusion signals in nuclei. The karyotype analysis showed hyperdiploidy as the most frequent abnormality. The frequency of patients with TEL/AML1 gene fusion was 19% (11 out of 58 cases). Six of the positive samples had normal karyotypes. Deletion of the wild-type TEL allele was observed in 27.3% of TEL/AML1 fusion-positive cases, but it was also identified in 4.2% of the negative cases. Three cases presented two fusion signals, indicating possible duplication of the der(21). The mean age of the patients with TEL/AML1 fusion was 4.8 years and the mean amount of peripheral leukocytes was 44,270 x 10(6)/L. The higher frequency of females with B-ALL (33/58 cases) observed in our sample was probably due to the selection mode of the study cases. The prevalence of TEL/AML1 fusion in Brazilian children in our study is similar to that found in other populations.

Restriction enzyme and fluorochrome banding analysis of the constitutive heterochromatin of Saguinus species (Callitrichidae, Primates).

Metaphases of Saguinas fuscicollis fuscicollis and Saguinas mystax were subjected to restriction enzyme banding (Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I and Msp I) and sequenced C-banding, together with fluorochrome staining (CMA3 and DAPI). Both species showed large C-bands in the pericentromeric regions. S. f. fuscicollis also manifested distal C-bands in both arms of pair 5 and in the short arms of pairs 8-15. In each species the heterochromatin revealed different reactions to the restriction enzymes and fluorochromes. This was related to its location in the genome (centromeric, pericentromeric, distal), making possible the identification of distinct categories of constitutive heterochromatin. In S. f. fuscicollis there were at least five types, namely centromeric in bi-armed chromosomes, centromeric in acrocentrics, pericentromeric, distal, and cryptic bands, detected only with the Alu I. There were three types in S. mystax, viz centromeric in bi-armed chromosomes, centromeric in acrocentric, and pericentromeric chromosomes. Several aspects of their constitution and origin are discussed.

Heterologous amplification of microsatellite loci from mouse and rat in oryzomyine and Thomasomyine South American rodents.

Eleven heterologous primers were investigated in 119 individuals of 11 species of rodents of the Oryzomyine and Thomasomyine groups. The animals were collected at four sites of the "Cerrado," a dryland biome located on the Brazilian Plateau, all of them being karyotyped and taxonomically allocated according to the karyotype. Four of these primers, R47, R65, R75 (from Rattus), and ATP (from Mus) cross-amplified in at least one of these taxa, giving products of seven, nine, one, and three bands, respectively. These values are of the same order as others obtained when heterologous primers were amplified in other orders of mammals. Of the 20 products amplified in these two rodent groups by these four primers, only 7 of the bands were seen in a heteromorphic state (one individual presenting two bands), in two species (Rhipidomys aff. leucodactylus and Oryzomys megacephalus). The others occurred as monomorphic bands.

Characterization of constitutive heterochromatin of Callithrix geoffroyi (Callitrichidae, Primates) by restriction enzymes and fluorochrome bands.

The neotropical primate genus Callithrix comprises two groups of species, jacchus and argentata, which inhabit distinct geographical regions and manifest different fur coloration and constitutive heterochromatin (CH) markers in their karyotypes. In this investigation the CH of a representative of the jacchus group, Callithrix geoffroyi, was analysed using fluorochromes and restriction enzymes in situ. To clarify the source of the constitutive heterochromatin of both groups, the data obtained in the jacchus group were compared with those published in the argentata group obtained by the same techniques. The C-bands of C. geoffroyi (four specimens, 2n = 46) were centromeric in all chromosomes, and distally located in pairs 6 and 22. The Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I, and Msp I restriction endonucleases and CMA3 and DAPI fluorochromes produced different bands, which allowed the characterization of four distinct types of constitutive heterochromatin in the C. geoffroyi genome. Several of these types of heterochromatin were present in the ancestor of the two groups of species, jacchus and argentata, while others originated after their cladogenesis.

(T2AG3)n telomeric sequence hybridization suggestive of centric fusion in karyotype marsupials evolution.

It has been suggested that the karyotype of the marsupials derived from a low diploid number (2n = 14) which originated, through fissions of biarmed chromosomes, the karyotypes with a higher 2n. The telomeric sequence (T2AG3)n was in situ hybridized to the chromosomes of Gracilinanus microtarsus and G. emiliae, Micoureus demerarae and Marmosa murina, species with 2n = 14, in Monodelphis sp., M. domestica, M. kunsi and M. brevicaudata with 2n = 18, and in Lutreolina crassicaudata, Didelphis albiventris, Chironectes minimus, Philander opossum and P. frenata, all of them with 2n = 22. The probe hybridization occurred in the telomeric regions of both arms, short and long, of all chromosomes of the complement of all individuals of all species analysed. However, in some pairs of the karyotypes of Gracilinanus microtarsus and Micoureus demerarae (with 2n = 14), and in Monodelphis sp., M. domestica, M. kunsi and M. brevicaudata (2n = 18) ectopic signs of hybridization were detected proximal to the centromeres, suggesting the retention of this telomeric sequence in the centromeric regions of some chromosomes of these species. Based on these results, it is proposed that the karyotype of marsupials evolved from a 2n = 22 to a 2n = 14, by means of chromosomal fusions.

The karyotype of the South American rodent Kunsia tomentosus (Lichtenstein, 1830).

Chromosomal analysis of Kunsia tomentosus showed a karyotype with 2n = 44, constituted by 21 pairs of acrocentric autosomes. The X chromosome was a median acrocentric, between pairs 3 and 4 in size, and the Y chromosome was a small acrocentric (between pairs 19 and 20). Five pairs with nucleolus organizer regions were located at the short arms. C-banding showed blocks of constitutive heterochromatin occurring in the centromeres of all autosomes and of the X chromosome. The Y chromosome was entirely heterochromatic. In order to identify possible homologies, karyotypes of Kunsia and Scapteromys, the phyletically related taxa, were compared. No autosome shared by either genus was found by G-band comparisons. The C-band patterns and those produced by Alu I, Mbo I, Rsa I and Hae III restriction endonucleases were also different. The results of FISH indicated a different composition of the telomeric regions of the chromosomes of both taxa, since in Scapteromys the probes hybridized in both telomeres, and in Kunsia this hybridization only occurred in one of the telomeres. These differences also occurred in the localization and number of nucleolus organizer regions.

Proposed chromosomal phylogeny for the South American primates of the Callitrichidae family (Platyrrhini).

Cytogenetic and cytotaxonomic studies (G, C, sequential G/C, and NOR banding) were performed on 110 specimens representing the four genera of South American primates of the family Callitrichidae: Cebuella (C. pygmaea), Callithrix, groups argentata (C. argentata, C. emiliae, C. chrysoleuca, C. humeralifera, C. mauesi), and jacchus (C. aurita, C. geoffroyi, C. jacchus, C. kuhli, C. penicillata), Leontopithecus (L. chrysomelas, L. rosalia), and Saguinus (S. midas midas, S. m. niger). Mitotic chromosomes are characterized, and the rearrangements distinguishing the karyotypes of the taxa are inferred from arm homologies. The results were then converted into numerical data and submitted to cladistic analysis. The following conclusions were achieved: 1) Five karyotypic classes were observed, which correspond to the five taxa studied. Differences between them are as follows: a) Cebuella (2n = 44, 10 acrocentrics, A + 32 bi-armed autosomes, bi) and the argentata group (2n = 44, 10A + 32bi) are different from each other due to a reciprocal translocation; b) both can be distinguished from the jacchus group (2n = 46, 14A + 30bi) by a centric fusion/fission rearrangement and a paracentric inversion; c) Leontopithecus (2n = 46, 14A + 30bi) and Saguinus (2n = 46, 14A + 30bi) differ from the jacchus group by a reciprocal translocation and three paracentric inversions; and d) Saguinus is different from the others by one paracentric inversion and pericentric inversions in at least four pairs of acrocentric autosomes. 2) The cladistic analysis separates Cebus (used as an outgroup) from the Callitrichidae groups, which forms a clade. Among the Callitrichidae, marmosets (Cebuella and Callithrix) form a sub-clade, Cebuella and the argentata group being more closely related to each other than both are to the jacchus group. Tamarins (Leontopithecus and Saguinus) are also quite close, so that if one was not derived from the other, they with the marmosets share a common ancestor. Among the tamarins, Leontopithecus is karyotypically closest to the marmosets, specifically to the jacchus group. 3) Based on the chromosome information and considering the possible direction of the evolutionary changes (primitivity or phyletic dwarfism hypothesis, previously advanced by other authors), it was possible to propose the ancestral karyotypes and to develop two alternatives for the origin, differentiation and dispersion of the callitrichid. Both proposals are plausible, but when the geographical distribution is considered, the phyletic dwarfism hypothesis seems to be the most probable.

Analysis of constitutive heterochromatin of Aotus (Cebidae, Primates) by restriction enzyme and fluorochrome bands.

The current classification of genus Aotus includes nine species, four of which occur above the Amazon River and five below it. The position of several of these taxa as a valid species has been questioned. Recently, we described the chromosomal constitution of a population in the state of Rondonia, Brazil, whose karyotype typically presented a considerable accumulation of constitutive heterochromatin. To best characterize these heterochromatins, in this work we subjected the metaphases of these animals to banding using AluI, HaeIII, HinfI, RsaI, DdeI, MboI and MspI restriction enzymes and CMA3 and DAPI fluorochromes. The banded metaphases were also submitted to sequential C-banding. RsaI, DdeI and MboI enzymes showed, in all chromosomes, a banding pattern of C type, similar to that obtained using barium hydroxide. This banding was also seen with AluI, HinfI and MspI, but with reduction or elimination of the C-bands in the chromosome pairs 1, 3-7 and 9. MspI also reduced the C-band of pairs 11, 16-21 and 23. HaeIII induced intermediate bands between G and C. Considering the data of the different bands produced, it was possible to characterize at least three distinct types of constitutive heterochromatin in Aotus from Rondonia: (a) centromeric bands, (b) bands of the heterochromatic short arms and (c) interstitial bands.

Chromosomal similarities and differences between tamarins, Leontopithecus and Saguinus (Platyrrhini, Primates).

The karyotypes of two taxa of genus Leontopithecus (rosalia and chrysomelas) are studied. Their G-, C- and NOR-banding patterns are compared with those of representatives of the genus Saguinus to determine chromosomal similarities and differences between the two genera and thus contribute to explaining phylogenetic relations between the tamarins. Leontopithecus, like the Saguinus, presents 2n = 46, 14 autosomes plus the Y acrocentric and 30 autosomes plus the X biarmed. No chromosomal rearrangement distinguishes the karyotypes of the representatives of genus Leontopithecus or genus Saguinus. The two genera are distinguished from each other by a paracentric inversion and pericentric inversions on at least four pairs of acrocentric autosomes, displacing the NORs of the small short arms in Leontopithecus to the proximal region of the long arms in Saguinus or vice versa. The tamarins are also distinguished by the distribution of noncentromeric constitutive heterochromatin. The data obtained indicate that the two tamarin genera are closely related chromosomally, suggesting that they probably originated from the same ancestral branch.

Comparative chromosomal study of five taxa of genus Callithrix, group jacchus (Platyrrhini, Primates).

The karyotypes of four marmoset species of the Callithrix jacchus group (C. aurita, C. kuhlii, C. geoffroyi, and C. penicillata) were investigated. The patterns of G-, C-, and NOR-bands of these karyotypes were compared with those of C. jacchus, previously described, in order to clarify the taxonomic relationships of this species group. All species present 2n = 46, 14 uni- and 30 biarmed autosomes, a median size submetacentric X chromosome, and the same NOR-band patterns. No rearrangement or constitutive heterochromatic variation differentiate these species, which differ only in the morphology of the Y chromosome. The data obtained indicate that, from the chromosomal point stand, the marmoset species of C. jacchus group constitute a homogeneous clade.

Analysis of constitutive heterochromatin by fluorochromes and in situ digestion with restriction enzymes in species of the group Callithrix argentata (Callitrichidae, Primates).

The karyotypes of the species belonging to the group Callithrix argentata (Callitrichidae, Platyrrhini) are characterized by large amounts of distal constitutive heterocharomatin (CH). The CH of the species C. argentata, C. humeralifera and C. emiliae was analyzed by banding with the restriction enzymes HinfI, MboI, aluI, RsaI, DdeI, HaeIII and MspI, as well as the fluorochromes CMA3 and DAPI. The results obtained permitted us to classify the CH of these species into three distinct types: 1) distal CH with a homogeneous response to enzymatic action, which was unchanged (HinfI, MboI, AluI, HaeIII), partially digested (DdeI) or fully digested (RsaI), being CMA3+, DAPI-; 2) centromeric CH, generally presenting a reduced band size. The varying extent of reduction, ranging from none to total, and also the variation of fluorochrome staining indicates that there is heterogeneity in this type of CH; 3) CH of the distal portion of the X chromosome of C. argentata and of the Y chromosome was CMA3- and unchanged by the enzymes, except for RsaI, which caused a reduction in size. MspI was the only enzyme unable to induce bands. Sequential C-banding permitted us to perceive banding variations that could not be observed simply by RE banding.

Chromosomal relationships and phylogenetic and clustering analyses on genes Callithrix group argentata (Callitrichidae, Primates).

The karyotypes of three species of marmosets of the Callithrix argentata group (C. argentata, C. humeralifera and C. chrysoleuca) were studied. Comparisons were made among species and with the previously described karyotypes of C. emiliae, C. mauesi (argentata group) and C. jacchus (jacchus group). Two chromosomes rearrangements differentiate the argentata (2n=44) and jacchus (2n=46) groups: fusion or fission and a paracentric invasion. The argentata group is also characterized by the addition of large amounts of distal constitutive heterochromatin (CH) in some chromosomes, while the jacchus group shows mainly centromeric heterochromatin. The five species of the argentata group differ in the amount or location of the distal CH. Interspecific differences were converted to a Basic Data Matrix (BDM), that was submitted to phenetic and cladistic analyses. For cladistic analyses C. jacchus was the outgroup. The results agree with morphological and geographical data.

Clastogenic effect of cisplatin on Wistar rat bone marrow cells.

The clastogenic effect of the drug cis-diamminedichloroplatinum II (cisplatin, CDDP) was investigated in Wistar rat bone marrow cells. Male rats, 3 per treatment time, aged 4 months and weighing 250-350 g were injected intraperitoneally with 6.0 mg/kg CDDP solution, and the control group received isotonic saline. The animals were sacrificed 6, 12, 18, 24 and 48 h after the injection. The chromosome preparation was obtained from bone marrow cells. Chromatid and chromosome aberrations were investigated in 300 metaphases per animal. A significant increase in number of chromosome aberrations was observed from 6 to 24 h, the majority being of the break and gap type. After 48 h a progressive reduction was observed, without differences from the negative control. These data confirm the mutagenic effect of CDDP in rats demonstrated for mice bone marrow by micronuclei assay, for murine ovary cells and mice spermatocytes.

Clastogenic effect of cisplatin on Wistar rat bone marrow cells

The clastogenic effect of the drug cis-diamminedichloroplatinum II (cisplatin, CDDP) was investigated in Wistar rat bone marrow cells. Male rats, 3 per treatment time, aged months and weighing 250-350 g were injected intraperitoneally with 6.0 mg/Kg CDDP solution, and the control group received isotonic saline. The animals were sacrificed 6, 12, 18, 24 and 48h after the injection. the chromosome preparation was obtained from bone marrow cells. Chromatid and chromosome aberrations were investigated in 300 metaphases per animal. A significant increase in number of chromosome aberration was observed from 6 to 24h, the majority being of the break and gap type. After 48 h a progressive reduction was observed, without differences from the negative control. These date confirm the mutagenic effect of CDDP in rats demonstrated for mice bone marrow by micronuclei assay, for murine ovary cells and mice spermatocytes

Distinct centric fusions in different populations of Deltamys kempi (Rodentia, Cricetidae) from South America.

Chromosome studies on 28 specimens collected in two Brazilian populations of the rodent species Deltamys kempi disclosed seven different karyotypes, due to two autosome centric fusions (2;3 and 9;15) in homozygous and heterozygous states, and a Y-autosome translocation present in all males. These results, plus those obtained previously in a population from Argentina and another from Brazil, show that this species has 44% of carriers of four autosome centric fusions, and each rearrangement is restricted to a distinct locality.

Densitometric measurements of C bands of chromosomes 1, 9, 16, and Y in leukemic and preleukemic disorders.

Fifty-six patients with blood disorders (23 with chronic myeloid leukemia, 14 with acute myeloblastic leukemia, seven with acute lymphoblastic leukemia, one with chronic lymphocytic leukemia, and 11 with preleukemia states) were studied. A quantitative and objective method of C band length analysis with well-matched controls was used. The C bands of chromosome pairs 1, 9, and 16 presented a normal distribution that was similar in patients and controls, whereas the Y chromosome presented an abnormal distribution. Smaller C bands in 1qh and higher indexes of intrapair heteromorphism in pairs 1 and 9 were detected in the CML group; the group of acute leukemias (myeloblastic and lymphoblastic) presented a smaller index only in pair 1qh. No other differences in length, heteromorphism, inversion frequency, or sex were detected.