Gianmarco Corneo

Repeated sequences in human DNA

Abstract Human DNA has been fractionated in Ag + Cs 2 SO 4 and Hg 2+ Cs 2 SO 4 preparative density gradients, and the fractions obtained have been centrifuged in neutral CsCl after extensive dialysis to eliminate Hg 2+ and Ag 2+ . By centrifugation in Ag + Cs 2 SO 4 a new satellite, called satellite DNA II, has been isolated from human DNA. It has a density of 1.693 g/ml. in neutral CsCl, accounts for 2% of the total approximately, renatures rapidly and separates into complementary strands having different densities in alkaline CsCl. In Hg 2+ Cs 2 SO 4 gradients human DNA appears to be composed of two classes of molecules. The first, which accounts for approximately 80% of the total, is highly heterogeneous in base composition, its density in CsCl ranging from 1.690 to 1.720 g/ml., and is distributed in Hg 2+ Cs 2 SO 4 so that the A·T-rich fractions are on the heavy side and the G·C-rich fractions on the light side, as expected on the basis of the preferential binding of Hg 2+ to A·T pairs. The second class, which accounts for approximately 15% of the total, is more homogeneous, has a density of 1.696 g/ml., and is located on the light side of the DNA band in the Hg 2+ Cs 2 SO 4 gradient. This suggests that the amount of Hg 2+ bound to this A·T-rich DNA is abnormally low. This second class of DNA has been isolated by preparative CsCl centrifugation from a pool of the light fractions obtained from DNA-Hg 2+ Cs 2 SO 4 centrifugation. It tends to renature after heat-denaturation, as shown by the shift of its density towards the native value in neutral CsCl.

The chromosomal location of human satellite DNA III

In situ hybridisation of radioactive complementary RNA has been used to localise the chromosomal distribution of human satellite DNA III. This DNA is found to be concentrated in paracentromeric heterochromatin mainly on chromosome 9 and in minor concentrations on chromosomes chiefly of the D and G groups.

Renaturation properties and localization in heterochromatin of human satellite DNA's

Abstract Human DNA has been fractionated by centrifugation in an Ag + -Cs 2 SO 4 preparative density gradient. Besides satellite DNA I and II, previously demonstrated and characterized, a newly identified satellite DNA III has been isolated, having a CsCl density of 1.696 g/ml and accounting for 1.5 % of the total genome. The renaturation properties of human satellite DNA III, estimated by determining its CsCl densities and melting curves after denaturation and renaturation, indicate that it is fast renaturing and therefore highly repeated, as are the other human satellite DNAs. The nuclei obtained from human placenta and leukemic leucocytes have been fractionated into heterochromatin and euchromatin. Satellite DNAs are enriched in heterochromatin, while they are no longer detectable in the DNA extracted from euchromatin, centrifuged in Ag + -Cs 2 SO 4 .

Gynaecomastia in men with chronic myeloid leukaemia after imatinib

cKit and platelet-derived growth-factor receptor (PDGFR) are receptor tyrosine kinases expressed in the testis, are involved in testosterone production, and are inhibited by imatinib. We measured hormone concentrations in 38 men receiving imatinib for chronic myeloid leukaemia at baseline and during treatment. Mean follow-up was 23.6 months (SD 7.5). We noted seven cases of gynaecomastia (18%, 95% CI 6-30%). A comparison of hormone concentrations in 21 patients before and during treatment showed that patients who developed gynaecomastia had a reduction in free testosterone concentrations of 29.53 pmol/L (95% CI 11.63-47.43), while patients who did not had a decrease of 6.36 pmol/L (-1.02 to 13.74). In most men with chronic myeloid leukaemia studied here, imatinib was associated with a reduction in the production of testicular hormones and in some, with the development of gynaecomastia.

Clustering of the DNA sequences complementary to repetitive nuclear RNA of HeLa cells.

Abstract We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.

Elution of human satellite DNAs on a methylated albumin kieselguhr chromatographic column:Isolation of satellite DNA IV

Abstract Human DNA has been eluted on a methylated albumin kieselguhr column: the fractions obtained have been centrifuged in Ag+-Cs2SO4 to show the presence of satellite DNAs. Satellite DNA II (density in CsCl = 1.693 g/ml) and the new satellite DNA IV (density in CsCl = 1.700 g/ml) are eluted early from the column and band, respectively, on the heavy side and the light side compared to the main DNA band in Ag+-Cs2SO4. Satellite DNA I (density in CsCl = 1.687 g/ml) and III (density in CsCl = 1.696 g/ml) are eluted late from the column. Satellite DNA IV separates into the complementary strands displaying a difference in density of 0.012 g/ml in alkaline CsCl and renatures rapidly after heat denaturation.

Human mitochondrial DNA

There is strong evidence that mitochondria once existed as free-living bacteria, which were taken up by primitive ancestors of eukaryotic cells. The host cell provided a ready source of energy-rich nutrients, and the mitochondrion provided a means to extract energy using oxygen. This attribute was key to survival, as oxygen accumulated in the primitive atmosphere. Mitochondria are physically in the same size range as bacteria, and the mt genome retains bacteria-like features. Like bacterial chromosomes and plasmids, the mt genome is a circular molecule. Also, very few noncoding sequences, or introns interrupt mt genes. These features are contrary to those of eukaryotic chromosomes, which are linear, and of eukaryotic genes, which have numerous introns.

Risk-oriented postremission strategies in adult acute lymphoblastic leukemia: prospective confirmation of anthracycline activity in standard-risk class and role of hematopoietic stem cell transplants in high-risk groups

INTRODUCTION Although definite risk classes are well known, risk-adapted modulation of first-line therapy is seldom attempted in adult ALL. So, a prospective validation of the therapeutic efficacy of a protocol (or a component thereof) in specific risk groups is uncommon. MATERIALS AND METHODS From 1996-1999 a risk-oriented program (08/96) was evaluated in 102/121 unselected patients (median age 35 years, blast count 0-450 x 10(9)/l, 100 B(lin) (lineage), 21 T(lin)) responsive to induction therapy. The standard risk (SR) class was B(lin) CD10+ Ph- with blasts < 10 x 10(9)/l (prior studies: disease-free survival (DFS) rate 52% at five years with dose-intensive anthracycline-containing programs). The SR protocol was therefore anthracycline-rich (early consolidation cycles with total idarubicin 96 mg/m2), and comprised long-term maintenance. High-risk (HR) patients were eligible to the following three options: allogeneic hematopoietic stem cell transplantation (HSCT) from related family donor; short sequence with high-dose cyclophosphamide-cytarabine-methotrexate followed by melphalan/total body irradiation with autologous HSCT; or T(lin) ALL chemotherapy regimen inclusive of high-dose cytarabine and methotrexate. RESULTS Treatment realization and three-year DFS rates according to risk class, HR subset and postremission treatment intensity were the following. SR group (n = 28): realization rate 93%, DFS 68.5%. HR group (n = 74): realization rate 80%, DFS 39% (P = 0.052 vs SR category). In HR group, three-year DFS rates by disease subtype were the following. B(lin) Ph- (n = 35) 43%; Ph+ (n = 19) 13% at 2.7 years (P = 0.006 vs other HR subtypes); T(lin) (n = 18) 59.5%. And DFS rates by treatment intensity were: allograft (n = 21) 40%; autograft (n = 28) 27%; shift to SR protocol (n = 13) 52% (P = ns vs allograft/autograft); T(lin) program (n = 10) 57%. Matched analyses of treatment protocols and disease subtypes suggested a possible therapeutic role of the autograft regimen in B(lin) Ph- ALL with a blast count < 25 x 10(9)/l, and of T(lin) protocol for T(lin) ALL. Comparisons with retrospective control cohorts were confirmatory of anthracycline activity in SR subclass. CONCLUSION The intended strategy was applicable to the majority of study patients, confirming the value of anthracyclines in SR class and, preliminarily, the usefulness a T(lin)-specific treatment. Apart from the case of Ph+ ALL, the indications for high-dose procedures with HSCT remains largely undetermined in this study.

Molecular organization and chromosomal location of human GC-rich heterochromatic blocks

From the sequencing of three genomic DNA fragments and PCR amplification products from total human DNA, we have derived the sequence of a 545-bp Sau3A fragment (68% GC), representative of a family of human DNA repeats. Since previous studies suggested its linkage with unrelated Sau3A repeats of 68 bp (54% GC) (beta-satellite sequences), this feature was further investigated by in situ hybridization experiments and by Southern blot analysis of a panel of DNAs from human-Chinese hamster somatic cell hybrids. Both DNA repeats are preferentially localized on the heterochromatic regions of acrocentric chromosomes, on the pericentromeric heterochromatin of chromosome 1, 3 and 9, and on the proximal euchromatic region of the chromosome Y q arm. On chromosome 9, both repeats are part of a 2.7-kb higher-order repeat unit. These results and the Southern blot analysis on partial digests of total DNA, suggest that the linkage between the two repetitive DNA sequences is a constant feature throughout the genome. Furthermore, Southern blot analysis of HpaII-digested and MspI-digested DNA from different human tissues and tumor cell lines indicates that the investigated heterochromatic blocks appear to be subjected to changes in their methylation pattern.

The organization of repeated DNA sequences in the human genome

The arrangement of repetitive and non-repetitive DNA sequences was studied in the human genome. By Ag+-Cs2SO4 density gradient centrifugations of human DNA at different fragment size reannealed to different C0t values and c-RNA hybridization experiments, we have shown the presence of two repetitive DNA fractions, called fast and slow intermediate DNA, with different pattern of sequence organization. — The fast intermediate DNA sequences (6% of the genome; CsCl density in renatured form: 1.703 g/ml) are in part clustered in fragments greater then 24,000 nucleotide pairs and in part in fragments ranging from 1,800 to 600 nucleotide pairs spaced with longer more complex sequences. — The slow intermediate DNA sequences (30% of the genome; CsCl density in renatured form: 1.707 g/ml) appear to be finely interspersed with non-repetitive sequences. At a DNA fragment size of 600 nucleotide pairs only a third of the slow intermediate DNA sequences are free of unique sequences, while the other two thirds are still organized with unique sequences. — It has also been shown that a great amount of the repetitive DNA sequence transcripts in heterogeneous nuclear RNA of HeLa cells are complementary to slow intermediate DNA sequences.