Manuel O. Diaz

A method for the rapid sequence-independent amplification of microdissected chromosomal material

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3 end and a defined sequence at its 5 end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.

Mapping of the shortest region of overlap of deletions of the short arm of chromosome 9 associated with human neoplasia.

Deletions of the short arm of chromosome 9 with a minimum region of overlap at band 9p22 are frequently observed in acute lymphoblastic leukemia and in gliomas. They also occur at a lower frequency in lymphomas, melanomas, lung cancers, and other solid tumors. These deletions often include the entire interferon (IFN) gene cluster, which comprises about 26 interferon-alpha (IFNA), -omega (IFNW), and-beta-1 (IFNB1) interferon genes, as well as the gene for the enzyme methylthioadenosine phosphorylase (MTAP). By comparing microscopic deletions with the genes lost at the molecular level, we have determined the order of these genes on 9p to be telomere-IFNB1-IFNA/IFNW cluster-MTAP-centromere. In a few cell lines and in primary leukemia cells, we have observed deletions that have breakpoints within the IFN gene cluster and result in partial loss of the IFN genes. These partial deletions allowed us to determine the order of some genes or groups of genes within the IFNA/IFNW gene cluster. Our current results map the shortest region of overlap of these deletions in the various tumors to the region between the centromeric end of the IFNA/IFNW gene cluster and the MTAP gene locus.

Sequence-independent amplification and labeling of yeast artificial chromosomes for fluorescence in situ hybridization

We have developed a method that allows reliable construction of high quality FISH probes from yeast artificial chromosomes (YACs) based on the separation of YACs by pulse-field gel electrophoresis and a rapid sequence-independent amplification procedure (SIA). These probes can be used to localize YACs on metaphase chromosomes and also with high efficiency, in interphase nuclei.

Giant readthrough transcription units at the histone loci on lampbrush chromosomes of the newt Notophthalmus.

We have studied transcription at the histone loci in oocytes of the newt Notophthalmus viridescens, using in situ hybridization of cloned probes to the nascent RNA on lampbrush chromosome loops. Clusters of the five histone coding sequences are separated by long tracts of a simple sequence DNA, satellite 1. We had previously demonstrated coordinate transcription of histone genes and satellite 1 sequences. We postulated that satellite sequences were transcribed by readthrough from histone gene promoters; that is, transcription initiated at any of the five usual promoters, but did not terminate at the 3′ end of the gene. Instead transcription proceeded through downstream sequences in the histone cluster (including spacers and downstream histone genes), and then through the satellite 1 region. Our model led to several specific predictions, in particular that some internal spacer regions between the genes should be well represented in the RNA on loops, that certain sequences should be absent from the loops, and that presence or absence of particular sequences should be correlated with morphological polarity of the transcription unit. We have hybridized ten strand-specific probes to the lampbrush chromosomes and we find that the patterns of hybridization agree with the readthrough model of transcription.

Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines.

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5 of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.

Hu-ets-2 is translocated to chromosome 8 in the t(8;21) in acute myelogenous leukemia☆

The human genome contains two distinct loci with homology to the viral ets gene, the transforming sequence of the E26 avian erythroblastosis virus; these loci, Hu-ets-1, and Hu-ets-2, have been mapped to 11q23 and 21q22, respectively. Using in situ chromosomal hybridization, we have demonstrated that Hu-ets-2 is translocated to chromosome #8, the chromosome containing the critical or conserved junction, as a result of the t(8;21) (q22;q22) in acute myelogenous leukemia. Another protooncogene, c-mos, is also retained at the conserved junction, suggesting that one or both of these genes may play a role in the pathogenesis of acute myelogenous leukemia.

Trisomy 8 in human hematologic neoplasia and the c-myc and c-mos oncogenes

The c-mos and c-myc proto-oncogenes have been assigned to bands q22 and q24, respectively, of human chromosome No. 8. A gain of chromosome No. 8 is the most common abnormality observed in myeloproliferative diseases. By using probes specific for the c-mos and c-myc genes, we have analysed the genomic DNA from peripheral blood and bone marrow samples from 15 patients with various malignant myeloid diseases, including leukemia and myelodysplasia, and from one patient with non-Hodgkins lymphoma, all of whom have trisomy for chromosome No. 8. Except for one patient, the c-mos and c-myc genes were found in restriction fragments of germline size. In one patient with myelodysplasia, one c-myc allele was rearranged in a Hind III fragment, the other allele being normal. Thus, trisomy 8 associated with human hematologic neoplasia is generally not related to gross rearrangements of the c-mos or c-myc genes.

DNA synthesis in the neo-X neo-Y sex determination system of Dichroplus bergi (Orthoptera: Acrididae)

DNA replication in the neo-X neo-Y sex determining system was studied by means of tritiated thymidine and autoradiography. Asynchronous replication was found in the X arm of the neo-X and the long arm of the neo-Y. In addition, striking asynchrony was also found for short isopycnotic homologous regions at the distal end of the autosmal arm of neo-X and the short arm of neo-Y to which pairing during meiosis is restricted. These short regions are asynchronous with respect to the heterochromatic segments as well as to the remaining proximal region of the autosomal euchromatic arm of neo-X. This difference in replication pattern within the same chromosome arm may be related to a differentiation between regions which are homozygous in both sexes and regions which are hemizygous in males.

Chromosomal sublocalization of the human p97 melanoma antigen

SummaryThe antigen p97 is a tumor-associated antigen that was first identified in human melanomas using monoclonal antibodies. Recently, p97 mRNA was purified and cloned, and a p97 cDNA clone was synthesized. By using the technique of in situ chromosomal hybridization, we have localized the p97 gene to human chromosome No. 3, at bands q28 to q29. p97 belongs to a superfamily of iron-binding proteins that have amino acid homology; other members of this family include transferrin (TF), lactotransferrin, and ovotransferrin. Based upon the shared amino acid homology and upon the observation that the nucleotide sequence is internally duplicated in these genes, it has been proposed that the TF superfamily arose from a common ancestral duplicated gene. The TF gene has also been mapped to the long arm of chromosome No. 3 at bands q21 to q23.

Analysis of tumor suppressor gene on human chromosome 9 in mouse x human somatic cell hybrids.

Deletions of the short arm of human chromosome 9 (9p) are common in human leukemia and solid tumors. The minimum region of overlap of these deletions, located between the interferon genes and the methylthioadenosine phosphorylase gene, is partially syntenic with a region of mouse chromosome 4 that has tumor suppressor activity. Somatic cell hybrids between tumorigenic, MTAP-deficient, mouse L cells, and MTAP-competent human cells containing either a normal copy of 9p or a 9p with a deletion involving band 9p21 were selected in culture conditions that require MTAP activity for continued growth. Somatic cell hybrids that contained a normal copy of 9p rarely formed tumors in nude mice. Cells from the rare tumors that grew had lost the normal 9p. Hybrid cells that contained a 9p with deletions formed tumors more frequently, and cells from these tumors retained the 9p deletion chromosome. These results provide evidence that a tumor suppressor gene (or genes) is located on human chromosome 9 within the region of deletion.