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N Sacchi

Publications and source records attributed to N Sacchi.

At least 19 recordsLinked to original sources
PubMed1994

AML1 fusion transcripts in t(3;21) positive leukemia: evidence of molecular heterogeneity and usage of splicing sites frequently involved in the generation of normal AML1 transcripts.

The t(3;21)(q26;q22) is associated with chronic myelogenous leukemia in blast crisis (CML-BC), leukemia evolving from (therapy-related) myelodysplasia, and with leukemia following other hematopoietic proliferative diseases. Molecular cytogenetic analysis and cloning of a few t(3;21) cases indicate that the breakpoints are quite heterogeneous even within a specific clinical phenotype. Interestingly some of the (3;21) breakpoints involve the AML1 gene previously found rearranged in the t(8;21) associated with acute myelogenous leukemia. AML1 is related to the Drosophila gene runt and is the human counterpart of the gene for the alpha subunit of the nuclear polyoma enhancer binding protein (PEBP2) also known as the core binding factor (CBF). In the t(3;21) AML1 was found rearranged with EAP, a gene on chromosome 3 encoding a small ribosomal protein, as well as with EV11, another gene on chromosome 3. Here we report our study of six cases of t(3;21). By using fluorescence in situ hybridization (FISH) analysis and AML1 probes we could conclude that at least in two CML-BC cases the breakpoint occurred in the AML1 intron that is disrupted by the t(8;21). An AML1/EAP fusion transcript, different from the one described in a therapy-related myelodysplasia, was detected in both CML-BC cases. This transcript is expected to result in a predicted protein containing the AML1 nuclear binding domain with an attached stretch of 17 amino acids unrelated to the EAP small ribosomal protein. In the other t(3;21) patients we could not detect an AML1/EAP transcript or an AML1/EV11 transcript. This result suggests heterogeneity of the t(3;21) at the molecular level. The AML1 chimeric transcripts identified so far, both in the t(3;21) and in the t(8;21), diverge from the normal transcripts either after exon 5 or exon 6. Here we show that in normal AML1 transcripts different splicing events are seen to occur after AML1 exon 5 as well as exon 6.

Amino Acid Sequence↗
PubMed1992

Transcriptionally active chimeric gene derived from the fusion of the AML1 gene and a novel gene on chromosome 8 in t(8;21) leukemic cells.

In the t(8;21)(q22;q22) of acute myelogenous leukemia (AML), the breakpoint on chromosome 21 disrupts the AML1 gene, generally in the intron between exons 5 and 6. To isolate fusion transcripts of AML1, and an as yet unidentified gene on chromosome 8 involved in the rearrangement, we used rapid amplification of cDNA ends (RACE) and primers for AML1 exons 5 and 6. A fusion transcript was identified by 3' RACE in the RNA of t(8;21) leukemic cells that also express multiple normal AML1 transcripts. This result clearly indicates that at least one transcriptionally active chimeric gene is generated by the chromosome translocation. This gene on the 8q- derivative represents the fusion between the 5' portion of the AML1 gene with the 3' portion of a chromosome 8 gene that contains a region of sequence homology with the cyclin D2 gene, here referred to as the CDR gene (cyclin D-related gene). The chimeric gene is probably responsible for the pathogenesis of the 8;21 AML. This finding makes it possible to detect the translocation at the molecular level, thus improving the diagnosis and monitoring of the disease in leukemic patients.

Base Sequence↗
PubMed1991

DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype. The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation. Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established. By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement. The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.

Base Sequence↗
PubMed1991

Single-cell detection of ets-1 transcripts in human neuroectodermal cells.

The genes of the ets family are thought to code for a novel class of transcriptional factors. These proteins have a specific DNA-binding domain different from the basic domain of both the helix-loop-helix and leucine zipper families of DNA-binding proteins. The ets-1 gene product has been shown to bind to the enhancer region of the human T-cell receptor alpha gene during thymocyte ontogeny. This finding explains the high expression of ets-1 observed in T cells and the correlation between ets-1 expression and the expression of the T-cell receptor gene during fetal development. The ets-1 gene is also possibly biologically active in neural cells. By using RNA in situ hybridization analysis, we demonstrate the presence of ets-1 transcripts in cells of peripheral embryonal neuroectodermal tumors, specifically neuroepithelioma and neuroblastoma. In addition, the gene is found transcribed in Ewing's sarcoma, postulated to be ontogenetically related to tumors derived from the neural crest.

Gene Expression↗
PubMed1990

ETS family of genes in leukemia and Down syndrome.

The human ETS2 and ERG genes are members of the ETS gene family, with sequence homology to the viral ets gene of the avian erythroblastosis retrovirus, E26. These genes are located on chromosome 21 and molecular genetic analysis of Down syndrome (DS) patients with partial trisomy 21 suggested that ETS2 may be a gene within the minimal DS genetic region. We have, in fact, been able to confirm the presence of the ETS2 gene dosage in triplicate occurring in occult human 21 chromosome abnormalities. It is known that ERG and ETS2 gene translocations occur in certain specific leukemias associated with defined chromosome rearrangements [e.g., t(8;21)]. Moreover, it is known that DS individuals are at greater risk for leukemic disease than their normal familial cohorts, implying that trisomy of that region of human chromosome 21 may play a role in the development of this type of neoplasia. The human ETS genes, first identified in our laboratory, are highly conserved, being found from lower organisms, like Drosophila and sea urchin, to humans. In mammals, the ETS genes are structurally distinct, located on separate chromosomes; they are transcriptionally active and differentially regulated. The ETS2 protein is phosphorylated and turns over with a half-life of approximately 20 min. After activation with the tumor promoter, TPA, the level of ETS2 elevates 5- to 20-fold. The properties of the ETS2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins thought to have regulatory functions in the nucleus. In the mouse thymus ets-1 and ets-2 are 8-10-fold higher, respectively, in the CD4+ subset than in other subsets examined, suggesting a role in T-cell development for these genes. Cells transfected with the cellular ets-2 gene, expressing higher levels of ets-2 products, showed a stimulated proliferation response, abolished their serum requirement and formed colonies in soft agar that could induce tumors in nude mice. Collectively, these data suggest that this family of genes might play a role in controlling specific steps of the signaling transduction pathway. Thus, the ETS genes, as other genes with homology to viral oncogenes, might be instrumental in regulating cellular growth and differentiation, as well as organismal development.

Animals↗
PubMed1990

Physical mapping of the human chromosome 11q23 region containing the ataxia-telangiectasia locus.

Two breakpoints within chromosome 11q23 were characterized with 29 DNA probes to establish a physical map of the region. This region is notable in that it contains at least 14 functional genes which are also syntenic in the mouse (chromosome 9). Chromosome 11q23 includes these markers: STMY, CLG, NCAM, DRD2, APOA1, APOC3, APOA4, CD3E, CD3D, CD3G, PBGD, THY1, ets-1, and cbl-2. The two breakpoints, herein called "X;11" and "4;11," defined a region of approximately 8 cM containing the APO and CD3 complexes as well as the polymorphic marker D11S29. DRD2 localized centromeric to the X;11 breakpoint despite evidence for close genetic linkage to D11S29, suggesting that DRD2 lies close to the X;11 breakpoint. THY1, PBGD, and cbl-2 localized telomeric to the 4;11 breakpoint and thus to the [D11S29--APO--CD3] grouping as well. The physical map helps to correlate the cytogenetic and linkage maps of this region. It also suggests that the human 11q23 syntenic grouping is inverted with respect to its murine counterpart. Based on this physical map and on our primary linkage map of the 11q23 region, we are able to confirm a preliminary localization of the gene for ataxia-telangiectasia group A (ATA) to a region centromeric to the interval defined by D11S144 (pYNB3.12) and THY1.

Animals↗
PubMed1990

The human Evi-1 gene is located on chromosome 3q24-q28 but is not rearranged in three cases of acute nonlymphocytic leukemias containing t(3;5)(q25;q34) translocations.

The murine Evi-1 gene encodes a protein that has multiple 28-amino acid repeats containing the consensus sequence found in the zinc finger domains of many transcriptional regulatory proteins. Activation of the expression of the Evi-1 gene is frequently found in murine myeloid leukemias and leukemia cell lines and is due to retroviral insertions in the 5' region of the gene in either the Evi-1 or the CB-1/FIM3 common sites of viral integrations. To examine the role of the Evi-1 gene in human leukemias we have cloned regions of the human locus corresponding to the coding region of the gene and regions corresponding to the Evi-1 and CB-1/FIM3 common sites of integrations. Using these probes we demonstrate that the human Evi-1 gene maps to chromosome 3q24-q28 in a region that is translocated in acute nonlymphocytic leukemias with a t(3;5)(q25;q34). By in situ hybridization with metaphase chromosomes from one patient with a 3;5 translocation, the Evi-1 gene was found to be translocated to the derivative 5 chromosome. However, no rearrangements were detected by Southern blot analysis with DNAs from three patients with a t(3;5) using probes from the Evi-1 or CB-1/FIM3 loci. No Evi-1 transcripts were detected with RNA from leukemic blasts of one patient with a t(3;5).

Blotting, Northern↗
PubMed1989

Kinetic studies of anthracycline-DNA interaction by fluorescence stopped flow confirm a complex association mechanism.

The kinetics of association and dissociation between calf thymus DNA and five anthracyclines, including doxorubicin, daunorubicin, and three synthetic analogues, were investigated with stopped flow using fluorescence detection. The sensitivity of this technique allowed us to work with submicromolar drug concentrations, thus excluding formation of aggregates, and with ratios of DNA base pairs to drug in the range 10-250, where site exclusion effects could be taken into account with a simple correction of DNA concentration and pseudo-first-order conditions were nearly fulfilled. In all cases, both association and dissociation reactions required a sum of three exponential terms to be fitted. However, satisfactory interpretation of reciprocal relaxation times as functions of DNA concentration was only achieved with kinetic models comprising a total of five steps. One of the extra steps was tentatively assigned to formation of a weakly bound, probably nonintercalated species. Another step was deduced from a comparison between results of association and dissociation experiments. The five steps are arranged, for convenience, in an association mechanism with two branches, though other mechanisms cannot be definitely ruled out. Correlation of cytotoxicity data with both association and dissociation rates is not found to be significant. This suggests that other factors must be involved in modulating the different biological properties of the investigated anthracyclines.

Animals↗
PubMed1989

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity.

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.

Antibiotics, Antineoplastic↗
PubMed1988

High efficiency in the attribution of parental origin of non-disjunction in trisomy 21 by both cytogenetic and molecular polymorphisms.

The precise origin of the supernumerary chromosome can be defined in the majority of trisomy 21 cases. This is achieved by evaluating the chromosome 21 short arm polymorphism and analysing restriction fragment length polymorphisms (RFLPs) of multiple chromosome 21 loci. We report a study on 37 Italian families with Down's syndrome. In 35 cases (94.6%) both the parental and the meiotic stage of non-disjunction could be established. Knowledge of the origin of the extra chromosome 21 is a pre-requisite for investigations of genetic or environmental factors that may affect the meiotic process.

Down Syndrome↗
PubMed1988

The ETS genes on chromosome 21 are distal to the breakpoint of the acute myelogenous leukemia translocation (8;21).

The definition of the genetic linkage map of human chromosomes may be helpful in the analysis of cancer-specific chromosome abnormalities. In the translocation (8;21)(q22;q22), a nonrandom cytogenetic abnormality of acute myelogenous leukemia (AML), we previously observed the transposition of the ETS2 gene located at the 21q22 region from chromosome 21 to chromosome 8. However, no ETS2 rearrangements were detected in the DNA of t(8;21)-positive AML cells. Genetic linkage analysis has allowed us to locate the ETS2 gene relative to other loci and to establish that the breakpoint is at an approximate genetic distance of 17 cM from ETS2. When the information from the linkage map is combined with that from molecular studies, it is apparent that (a) the t(8;21) breakpoint does not affect the ETS2 gene structure or the structure of the other four loci proximal to ETS2: D21S55, D21S57, D21S17, and ERG, and ETS-related gene; and (b) the actual DNA sequence involved in the t(8;21) must reside in a 3-cM genetic region between the D21S58 and the D21S55/D21S57 loci, and remains to be identified.

Blotting, Northern↗
PubMed1988

Lack of evidence for association of meiotic nondisjunction with particular DNA haplotypes on chromosome 21.

The hypothesis of a predisposition to meiotic nondisjunction for chromosome 21 carrying a specific molecular haplotype has been tested. The haplotype in question is defined by the restriction fragment length polymorphisms for the D21S1/D21S11 loci. Our results obtained on a sample of Northern Italian families with the occurrence of trisomy 21 (Down syndrome) failed to support this hypothesis, contradicting a previous study [Antonarakis, S. E., Kittur, S. D., Metaxotou, C., Watkins, P. C. & Patel, A. S. (1985) Proc. Natl. Acad. Sci. USA 82, 3360-3364]. These findings rule out an association between any specific D21S1/D21S11 haplotype (as well as other haplotypes for the D21S13, ETS2, and D21S23 loci) and a putative cis-acting genetic element favoring the meiotic missegregation of chromosome 21. For this reason, no preventive screening for couples at risk for trisomy 21 may be based on any of the haplotypes tested.

Adult↗
PubMed1988

Comparative mapping of DNA markers from the familial Alzheimer disease and Down syndrome regions of human chromosome 21 to mouse chromosomes 16 and 17.

Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid beta precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, we have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS.

Alzheimer Disease↗