Letizia Longo

The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly understood. We have tested the notion that the chromosome make-up may be important in contributing both to somatic cell chimaerism and to germ line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percentage of euploid metaphases in the ES cell clones injected into the murine blastocyst. The majority of the ES cell clones that we tested, which were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in contrast, none of the ES cell clones with more than 50% of chromosomally abnormal metaphases transmitted to the germline. Euploid ES cell clones cultured in vitro for more than 20 passages rapidly became severely aneuploid, and again this correlated closely with the percentage of chimaerism and with the number of ES cell--embryo chimaeras obtained per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the proportion of euploid cells dropped below 50%. This study suggests that aneuploidy, rather than ‘loss of totipotency’, in ES cells, is the major cause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of germline transmission, karyotype analysis is crucial and time/cost saving in any gene-targeting experiment

Frequency of RAS and p53 mutations in acute promyelocytic leukemias

The frequency of RAS and p53 mutations was investigated in 30 acute promyelocytic leukemias by single strand conformation polymorphism analysis and direct sequencing of genomic DNA. Only two cases bore N-RAS codon 12 mutations and none had p53 mutations responsible for aminoacid substitutions. It would, therefore, seem that neither RAS nor p53 are involved in acute promyelocytic leukemogenesis.

Structural and functional organization of the HF.10 human zinc finger gene (ZNF35) located on chromosome 3p21–p22

We report the structural and functional characterization of the HF.10 zinc finger gene (ZNF35) in normal human cells, as well as a processed pseudogene. The HF.10 gene spans about 13 kb and it is interrupted by three introns. All 11 zinc finger DNA-binding domains are contiguously encoded within the last 3 exon. The genomic region surrounding HF.10 exon 1 contains a CpG island and acts as a promoter in vitro. Using transient CAT assay in cotransfection experiments in cultured cells, we have determined that the HF.10 finger protein is a transcriptional transactivator. Restriction enzyme mapping and partial nucleotide sequencing of the HF.10 pseudogene indicated that it has arisen by retroposition of spliced HF.10 mRNA. In situ hybridization experiments revealed that both the functional locus and the pseudogene map to chromosome 3p21p22, a region that is frequently deleted in small cell lung and renal carcinomas. Hybridization of the HF.10 gene and the HF.10 pseudogene DNA probes to metaphases from a small cell lung carcinoma cell line with the 3p deletion revealed that both loci are part of the deleted chromosome region.

Molecular Genetics of the t(15;17) Translocation in Acute Promyelocytic Leukemia

Acute Promyelocytic Leukemia (APL) is a distinct subset of acute myeloid leukemia (AML) (FAB-M3 according to the French-American-British Cooperative Group). It is morphologically distinguished by the presence of hyper-granular leukemic blasts (Sultan et al. 1973; Bennett et al. 1976). The clinical propensity of APL patients to hemorrhage, the feature that first drew the attention to this type of acute leukemia, is thought to be due to the release of procoagulants by the granules in the malignant promyelocytes (Gralnick and Abrell 1973; Falanga et al. 1988). APL are cytogenetically distinguished by a balanced reciprocal chromosome 15; 17 translocation- t(15;17) (Rowley et al. 1977; Mitelman 1988). The high frequency (70 to 100% of cases) and the tumor-type specificity of the t(15;17) suggest that chromosome break-points affect DNA sequence that might be crucial in the pathogenesis of this malignancy.