Understanding the crystallographic and nanomechanical properties of bryozoans.

This study examines how microscale differences in skeletal ultrastructure affect the crystallographic and nanomechanical properties of two related bryozoan species: (i) Hornera currieae, which is found at relatively quiescent depths of c. 1000 m, and (ii) Hornera robusta, which lives at depths of 50-400 m where it is exposed to currents and storm waves. Microstructural and Electron Backscatter Diffraction (EBSD) observations show that in both species the secondary walls are composed of low-Mg calcite crystallites that grow with their c-axes perpendicular to the wall. Branches in H. currieae develop a strong preferred orientation of the calcite c-axes, while in H. robusta the c-axes are more scattered. Microstructural observations suggest that the degree of scattering is controlled by the underlying morphology of the skeletons: in H. currieae the laminated branch walls are smooth and relatively uninterrupted, whereas the wall architecture of H. robusta is modified by numerous deflections, forming pustules and ridges associated with microscopic tubules. Modelling of the Young's modulus and measurements of nanoindentation hardness indicate that the observed scattering of the crystallite c-axes affects the elastic modulus and nanohardness of the branches, and therefore controls the mechanical properties of the skeletal walls. At relatively high pressure in deep waters, the anisotropic skeletal architecture of H. currieae is aimed at concentrating elasticity normal to the skeleton wall. In comparison, in the relatively shallow and active hydrographic regime of the continental shelf, the elastically isotropic skeleton of H. robusta is designed to increase protection from external predators and stronger omni-directional currents.

Life table parameters of the red palm mite Raoiella indica (Acari: Tenuipalpidae) at various temperatures and for sexual and asexual reproduction.

The red palm mite, Raoiella indica Hirst, is a threat to coconut, banana and native Arecaceae and Heliconiaceae in Brazil. This mite originated in the Eastern Hemisphere and was first reported in 2004 in the Americas, where the pest is spreading quickly and causing severe damage to its host plants. The objective of this work was to determine the life-history parameters of R. indica at constant temperatures, estimate its thermal requirements [threshold temperature (Tb) and thermal constant (K)] and also compare its life table parameters between sexual reproduction and parthenogenesis. The life tables were constructed on leaflets of Adonidia merrillii at 15, 20, 24, 27, 30 and 34 °C and 65% RH and a 12-h photoperiod. The longevity and the number of laid eggs of non-copulated adult females were evaluated at 27 °C. Raoiella indica had complete development, from egg to adult, only at 20, 24, 27 and 30 °C. At 15 °C, the eggs did not hatch, and at 34 °C, the mites survived only until the larval stage. For sexual reproduction, the optimal temperature was 27 °C, under which the reproductive parameters were higher. The reproductive parameters for sexual reproduction were higher than those for parthenogenesis. The Tb was 14.79 °C, and the thermal constant was 208.33 degree days. The life parameters estimated in this study can be used for modelling and predicting the population growth of R. indica in the field and consequently for improving their management strategies.

Characterisation of functional deficits induced by AAV overexpression of alpha-synuclein in rats.

Background: In the last decades different preclinical animal models of Parkinson's disease (PD) have been generated, aiming to mimic the progressive neuronal loss of midbrain dopaminergic (DA) cells as well as motor and non-motor impairment. Among all the available models, AAV-based models of human alpha-synuclein (h-aSYN) overexpression are promising tools for investigation of disease progression and therapeutic interventions. Objectives: The goal with this work was to characterise the impairment in motor and non-motor domains following nigrostriatal overexpression of h-aSYN and correlate the behavioural deficits with histological assessment of associated pathology. Methods: Intranigral injection of an AAV9 expressing h-aSYN was compared with untreated animals, 6-OHDA and AAV9 expressing either no transgene or GFP. The animals were assessed on a series of simple and complex behavioural tasks probing motor and non-motor domains. Post-mortem neuropathology was analysed using immunohistochemical methods. Results: Overexpression of h-aSYN led to progressive degeneration of DA neurons of the SN and axonal terminals in the striatum (STR). We observed extensive nigral and striatal pathology, resembling that of human PD brain, as well as the development of stable progressive deficit in simple motor tasks and in non-motor domains such as deficits in motivation and lateralised neglect. Conclusions: In the present work we characterized a rat model of PD that closely resembles human PD pathology at the histological and behavioural level. The correlation of cell loss with behavioural performance enables the selection of rats which can be used in neuroprotective or neurorestorative therapies.

Lateralized deficits after unilateral AAV-vector based overexpression of alpha-synuclein in the midbrain of rats on drug-free behavioral tests.

BACKGROUND: Preclinical rodent models of Parkinson's aim to recapitulate some of the hallmarks of the disease as it presents in humans, including the progressive neuronal loss of dopaminergic neurons in the midbrain as well as the development of a behavioral phenotype. AAV vector-based models of alpha-synuclein overexpression are a promising tool to achieve such animal models with high face and predictive validity. OBJECTIVE: We have developed a preclinical rodent model of Parkinson's disease using an AAV-vector based overexpression of human alpha-synuclein. In the present work we characterize this model on a behavioral and histopathological level. METHODS: We use a AAV9 vector for transgene delivery to overexpress human alpha-synuclein under a CBA promoter. We compare the behavioral and histopathological changes to a AAV vector control group where the transgene was omitted and to that of a 6-OHDA lesion control. We assessed the behavioral performance of these three groups on a series of tests (Cylinder, Stepping, Corridor) at baseline and up to 22 weeks post-injection at which point we performed electrochemical recordings of dopamine kinetics. RESULTS: The overexpression of human alpha-synuclein led to the progressive manifestation of behavioral deficits on all three behavioral tests. This was accompanied with impaired dopamine release and reuptake kinetics as demonstrated by electrochemical detection methods. Histopathological quantifications corroborated the findings that we induced a moderate cell loss with remaining cells displaying pathological markers which are abundant in the brains of human PD patients. CONCLUSIONS: In the present work we developed a characterized a rat model of PD that closely mimics human disease development and pathology. Such model will be of great use for investigation of disease mechanisms and early therapeutic interventions.

Associations of risk factors obesity and occupational airborne exposures with CDKN2A/p16 aberrant DNA methylation in esophageal cancer patients.

It is known that obesity and occupational airborne exposure such as dust are among risk factors of esophageal cancer development, in particular squamous cell carcinoma (SCC) of esophagus. Here, we tested whether these factors could also affect aberrant DNA methylation. DNAs from 44 fresh tumor tissues and 19 non-tumor adjacent normal tissues, obtained from 44 patients affected by SCC of esophagus (SCCE), were studied for methylation at the CDKN2A/p16 gene promoter by methylation-specific polymerase chain reaction assay. Statistical methods were used to assess association of promoter methylation with biopathological, clinical, and personal information data, including obesity and airborne exposures. Methylation at the CDKN2A/p16 gene promoter was detected in 12 out of 44 tumor samples. None of the non-tumor tissues exhibited the aberrant methylation. Our results confirmed previously described significant association with low tumor stage (P= 0.002); in addition, we found that obesity (P= 0.001) and occupational exposure (P= 0.008) were both significantly associated with CDKN2A/p16 promoter methylation. This study provides evidence that obesity and occupational exposure increase the risk of developing esophageal cancer through an enhancement of CDKN2A/p16 promoter methylation.

Geographical Distribution of Schizotetranychus hindustanicus and Associated Mites in Roraima, Brazil.

The Hindustan citrus mite, Schizotetranychus hindustanicus Hirst (Acari: Tetranychidae), is a quarantine pest present in the state of Roraima, Brazil. This mite, which was described in India in 1924, was reported in 2002 in Venezuela and spread to Roraima, where it was reported in 2008, and to Colombia, where it was reported in 2010. Its possible spread to other regions of Brazil is a threat to Brazilian citriculture. This study reports the current distribution of S. hindustanicus and potential predators of this pest and other mites associated with citrus in Roraima. A survey was conducted in August and September 2015 in the 15 municipalities of the state. In each municipality, orchards and citrus plants in backyards and public areas along highways and in urban areas were sampled. Samples of leaves and fruits were collected to identify the mite and its natural enemies. Schizotetranychus hindustanicus was found in all 15 municipalities in the state of Roraima. In total, 308 associated mites were found, with S. hindustanicus being the most abundant phytophagous mite, followed by Brevipalpus yothersi Baker. Amblyseius aerialis (Muma) was the most abundant predator, followed by Iphiseiodes zuluagai Denmark and Muma and Euseius concordis (Chant). The broad dispersal of S. hindustanicus in Roraima increases the risks of this pest reaching the main citrus-producing regions in Brazil.

Heart rate reduction with ivabradine in the early phase of atherosclerosis is protective in the endothelium of ApoE-deficient mice.

Ivabradine, a heart rate reducing agent, protects the vascular system by unidentified mechanisms. We sought to determine the effects of the treatment with ivabradine, started before plaque formation, on early transcriptional changes and endothelium lesions in regions of aorta subjected to disturbed blood flow. Six week-old apolipoprotein E-deficient (ApoE-/-) mice, fed a low-fat diet, were treated with ivabradine to determine the effect on transcriptional changes (2-and 4-week treatment) and on lesions formation (19-week treatment) in the endothelium of the aortic arch. Microarrays analysis (60k probes) of endothelium-enriched RNA was carried out to detect changes in gene expression induced by treatment. Endothelium damage was assessed by en-face immunofluorescence staining for vascular endothelial (VE) cadherin. According to microarray analysis, 930 transcripts were affected by the treatment. We found downregulation of pro-apoptotic and pro-inflammatory genes, the majority of which are nuclear factor-κB (NF-κB)-and/or angiotensin II-regulated genes, and upregulation of anti-inflammatory genes. Many shear stress-responsive genes were affected by the treatment and the MAPK, Notch signalling and sterol metabolic processes were among the most significantly affected pathways. Consistently, we observed increased levels of Hes5, a Notch target gene, together with a reduction of endothelium damage, in the lower aortic arch of treated- compared with untreated- mice. We concluded that an early treatment with ivabradine protected the endothelium of the aortic arch of ApoE-/- mice. Activation of the Notch signalling could be part of the mechanism underlying this protection.

Anticancer activity of an adenoviral vector expressing short hairpin RNA against BK virus T-ag.

The human polyomavirus BK (BKV) is oncogenic in rodents and induces malignant transformation of rodent cells in vitro. Although its role in human tumorigenesis is still debated, BKV represents an excellent model to evaluate molecularly targeted antineoplastic approaches. Here, we have tested whether stable suppression of the T antigen (T-ag) oncogene expression could inhibit the in vitro and in vivo malignant phenotype of BKV-transformed mouse cells. An adenovirus vector system that expresses small hairpin RNAs (shRNAs), which are converted into active small interfering RNAs (siRNA) molecules against the BKV T-ag, was developed. This vector was able to inhibit the expression of BKV T-ag through a highly efficient in vitro and in vivo delivery of the siRNA molecule. In addition, it allowed a stable expression of siRNA for a period of time sufficient to elicit a biological effect. Inhibition of T-ag expression results in reduction of the in vitro growth rate of BKV-transformed cells, which is, at least in part, caused by restoration of p53 activity and induction of apoptosis. In vivo studies proved that adenovirus vectors expressing anti-T-ag siRNA were able to suppress tumorigenicity of BKV-transformed cells. Moreover, adenovirus vector direct treatment of growing tumors resulted in a significant reduction of tumor growth. This study indicates that siRNAs delivery via a viral vector have a potential usefulness as in vivo anticancer tool against viral and cellular oncogenes.

BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.

Anti-leukemic activity of microRNA-26a in a chronic lymphocytic leukemia mouse model.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eµ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

MicroRNA deregulation in human thyroid papillary carcinomas.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in a wide range of basic processes such as cell proliferation, development, apoptosis and stress response. It has recently been found that they are also abnormally expressed in many types of human cancer. We analyzed the genome-wide miRNA expression profile in human thyroid papillary carcinomas (PTCs) using a microarray (miRNACHIP microarray) containing hundreds of human precursor and mature miRNA oligonucleotide probes. Using this approach, we found an aberrant miRNA expression profile that clearly differentiates PTCs from normal thyroid tissues. In particular, a significant increase in miRNA (miR)-221, -222 and -181b was detected in PTCs in comparison with normal thyroid tissue. These results were further confirmed by northern blot and quantitative RT-PCR analyses. Moreover, RT-PCR revealed miR-221, -222 and -181b overexpression in fine needle aspiration biopsies corresponding to thyroid nodules, which were eventually diagnosed as papillary carcinomas after surgery. Finally, miR-221, -222 and -181b overexpression was also demonstrated in transformed rat thyroid cell lines and in mouse models of thyroid carcinogenesis. Functional studies, performed by blocking miR-221 function and by overexpressing miR-221 in human PTC-derived cell lines, suggest a critical role of miR-221 overexpression in thyroid carcinogenesis. In conclusion, these data, taken together, indicate an miRNA signature associated with PTCs, and suggest miRNA deregulation as an important event in thyroid cell transformation.

Genomic organization of the ATM locus involved in ataxia-telangiectasia.

The ATM gene, involved in the genetic disorder ataxia-telangiectasia (AT), has been identified recently. This gene is suspected to predispose to malignancy and is located in a chromosomal region that we have recently found deleted in 50 to 60% of breast and lung carcinomas. Because of its location and its function, the ATM gene is a strong candidate tumor suppressor or modifier gene of chromosome region 11q23. In this study, we define its genomic structure. The aim was to establish the basis for the development of mutation scanning methods based on DNA instead of RNA. We found that the gene spans a region of approximately 70-80 kb and is composed of 37 exons, ranging in size from 64 to 324 bp. Nucleotide sequences of all exon/intron boundaries were determined. With this information, it will be possible to develop simple genetic tests for the identification of homozygotes and heterozygotes, as well as determine whether the gene is involved in the pathogenesis of breast and other carcinomas.

The coding region of the Bloom syndrome BLM gene and of the CBL proto-oncogene is mutated in genetically unstable sporadic gastrointestinal tumors.

Microsatellite instability (MSI) characterizes the hereditary nonpolyposis colorectal cancer syndrome but is also found in sporadic tumors. Frameshifts in microsatellites found in the coding regions (CDRs) of the TGFbeta1-RII, IGFIIR, hMSH3, hMSH6, and BAX genes indicate that MSI is involved in tumorigenesis by targeting genes that are directly implicated in the tumorigenic process. To identify additional genes targeted for MSI, we performed an analysis of the GenBank database that revealed 21 microsatellite repeats located in the CDR of 18 genes (12% of the analyzed sequences) whose function could be potentially associated with the tumorigenic process. Mutational studies of 57 sporadic gastrointestinal tumor DNAs revealed the presence of length variations in three of them: (a) BLM; (b) CBL; and (c) HOXA1. In the BLM gene, we found a frameshift mutation in a polyadenine repeat, whereas in the CBL proto-oncogene, an expansion of a trinucleotide repeat was detected with no translation shift. These alterations were present in 18 and 9%, respectively, of the genetically unstable sporadic gastrointestinal tumors analyzed, but in none of the cancers without the mutator phenotype. These changes were present in the DNA from the tumor but not in that from normal cells of the same patient. The HOXA1 retraction of a trinucleotide repeat was as frequent in both types of cancers and was also found in some normal paired tissues, therefore behaving as a neutral polymorphism. Our data extend the spectrum of unstable microsatellites located in gene CDRs and suggest that BLM and possibly CBL are involved in gastrointestinal tumorigenesis. Based on its proposed function, the BLM gene could represent a link between MSI and chromosomal instability pathways, because MSI targeting of the BLM gene could generate hypermutability and/or chromosomal instability.

Down-regulation of bcl-2 by p53 in breast cancer cells.

bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins. The result suggested that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down-regulate bcl-2 expression. We found, indeed, that overexpression of a mutant p53 (mut 175) in MCF-7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory element responsible for the down-regulation. If this mechanism will be proved also for the wild-type p53 allele, it may disclose a possible mechanism for p53-induced apoptosis: down-regulation of bcl-2.

GOK: a gene at 11p15 involved in rhabdomyosarcoma and rhabdoid tumor development.

The expression of GOK, a gene recently identified at 11p15.5, was studied in breast cancer, rhabdomyosarcoma, and rhabdoid tumor cell lines. In these neoplasms, deletions at 11p15 and suppression of tumorigenicity induced by a normal human chromosome 11 were previously demonstrated. Whereas breast cancer cell lines express readily detectable levels of GOK mRNA, expression is absent in rhabdomyosarcoma and rhabdoid tumor cell lines. This is in contrast with the high expression of GOK in skeletal muscle, the normal tissue of origin of rhabdomyosarcomas, suggesting that down-regulation of GOK expression could be involved in tumor development. In agreement with this hypothesis, transfection of GOK cDNA into G401 derived from a rhabdoid tumor and RD cells derived from a rhabdomyosarcoma that do not express detectable levels of GOK mRNA, induced cell death. Because GOK expression is not compatible with growth of these tumor cells, these results support the hypothesis that loss of GOK expression plays a role in tumor establishment or progression and suggest that GOK may act as a recessive tumor suppressor gene in rhabdomyosarcomas and rhabdoid tumors. On the contrary, transfection of GOK cDNA into the breast cancer cell line HBL100 produced no detectable effects, indicating that the growth-suppressive effect of GOK in RD and G401 cells was specific. Because rhabdomyosarcomas have been observed in cases of Beckwith-Wiedemann syndrome, a genetic disorder linked to 11p15, a role of GOK in this disease cannot be excluded.

Loss of heterozygosity at chromosome 11q in lung adenocarcinoma: identification of three independent regions.

We examined the pattern of allelic loss in 76 adenocarcinomas of the lung using 14 highly informative microsatellite markers on the long arm of chromosome 11. Loss of heterozygosity was found in 48 of 76 tumors (63%). Three distinct regions of deletion were identified. The first region, the most centromeric, lies between markers D11S940 and CD3D: the second, delimited by markers D11S924 and D11S925, is estimated to be 4 Mb in length, and has never been previously described; a third, more telomeric region, the length of which is also estimated to be in the range of 4 Mb, is bracketed by loci D11S1345 and D11S1328. These findings suggest the presence of at least three tumor suppressor genes on the long arm of chromosome 11, and confirm the relevance of 11q22-24, a region frequently deleted in carcinomas of the breast, ovary, uterine, cervix, colon, and malignant melanoma in the pathogenesis of solid tumors. The characterization of minimal regions of loss could provide the basis for the identification and cloning of the critical genes.

The t(6;16)(p21;q22) chromosome translocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene.

Very little is known about the molecular and genetic mechanisms involved in prostate cancer. Previous studies have shown frequent loss of heterozygosity (40%) at chromosomal regions 8p, 10q, and 16q, suggesting the presence of tumor suppressor genes in these regions. The LNCaP cell line, established from a metastatic lesion of human prostatic adenocarcinoma, carries a t(6;16)(p21;q22) translocation. To determine whether this translocation involved genes important in the process of malignant transformation, we cloned and sequenced the t(6;16) breakpoint of this cell line. Sequence analysis showed that the breakpoint is within the haptoglobin gene cluster on chromosome 16, and that, on chromosome 6, the break occurs within a novel gene, tpc, similar to the prokaryotic S10 ribosomal protein gene. The translocation results in the production of a fusion transcript, tpc/hpr.

Complete exon structure of the ALL1 gene.

The ALL1 gene is found rearranged in approximately 10% of acute lymphoblastic leukemias and in over 5% of acute myeloid leukemias. The gene undergoes fusion with either a variety of partner genes located on different chromosomes or with itself. To further characterize the role of the ALL1 gene in the leukemogenic process, and possibly in solid malignancies, we defined its complete genomic structure. The gene, which spans a region on chromosome band 11q23 approximately 90 kb in length, consists of 36 exons, ranging in size from 65 bp to 4249 bp. The determination of intronic sequences flanking the exon boundaries will allow the determination of whether point mutations may be responsible for inactivation of the gene in solid tumors showing loss of heterozygosity at region 11q23.

Common region of ALL-1 gene disrupted in epipodophyllotoxin-related secondary acute myeloid leukemia.

Translocations at chromosomal band 11q23 characterize most de novo acute lymphoblastic leukemias (ALL) of infants, acute myeloid leukemias (AML) of infants and young children, and secondary AMLs following epipodophyllotoxin exposure. The chromosomal breakpoints at 11q23 have been cloned from isolated cases of de novo ALL and AML. Using an 859-base pair BamHI fragment of human ALL-1 complementary DNA that recognizes the genomic breakpoint region for de novo ALL and AML, we investigated two cases of secondary AML that followed etoposide-treated primary B-lineage ALL. In the first case, the translocation occurred between chromosomes 9 and 11 and the breakpoint at 11q23 localized to the same 9-kilobase region of the ALL-1 gene that is disrupted in most of the de novo leukemias. In the second case the translocation was between chromosomes 11 and 19. The breakpoint occurred outside of the ALL-1 breakpoint cluster region.