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Summary Repetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease., Graphical Abstract Highlights ► In healthy subjects, the FSHD locus is a Polycomb repressive target ► FSHD patients display loss of Polycomb silencing and gain of Trithorax activation ► DBE-T is a chromatin-bound ncRNA expressed selectively in FSHD patients ► DBE-T recruits Ash1L to the FSHD locus to coordinate long-range gene de-repression, Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant myopathy associated with a reduced number of D4Z4 repeats, which leads to the expression of a noncoding RNA in a nearby locus. This RNA recruits a Trithorax epigenetic activator, which then switches on the transcription of genes driving FSHD.

The constitutive over-expression of the retinol dehydrogenase 10 (RDH10) gene, involved in retinoic acid (RA) biosynthesis, produced in HepG2 cells a significant antiproliferative response, but not signs of apoptosis. An indirect assay based on the Chloramphenicol AcetylTransferase (CAT) reporter gene driven by a retinoic acid responsive element (RARE) suggests in genetically modified HepG2 cells an increase of the endogenous RA concentration. Furthermore, the growth arrest of HepG2 cells over-expressing the RDH10 gene was associated with the upregulation and downregulation of, respectively, RARbeta/p21(Cip1) and CycE/CdK2 mRNAs. These results indicated that forced expression of RDH10 produces antiproliferative effects highly comparable to those achieved by retinoids treatment and thus the development of a gene therapy, finalized at the restoration of the enzymatic and receptorial machinery of the RA pathway, could be a possible curative strategy for hepatocellular carcinoma (HCC).

In Macaca mulatta, the single rDNA array is flanked by a patchwork of sequences including subregions of human Yp11.2, 4q35.2, and 10p15.3. This composite DNA region is characterized by unique or low-copy sequences, resembling a potentially transcribed region. The analysis of Cercopithecus aethiops, Presbytis cristata, and Hylobates lar suggests that this complex sequence organization could be shared by Old World monkey and lesser ape species. After the lesser apes/great apes divergence, the unique or nonduplicated DNA region underwent amplification and spreading, preferentially marking the p arm of acrocentric chromosomes bearing the rDNA. The molecular analysis of human acrocentric chromosomes revealed some extent of remodeling of the rDNA boundary: near the human NOR, a large 4q35.2 duplication partially resembles that found in MMU; conversely, infrequently represented Yp11.2 sequences totally differed from those of the macaque, and 10p15.3 sequences were lacking. Thus, although evolutionary events modified the sequence organization of the MMU rDNA boundary, its overall sequence feature and the preferential location in vicinity to the NOR have been conserved. © 2006 Elsevier Inc. All rights reserved.

To investigate the molecular events taking place during the development of hydraulic interstitial edema, we analyzed by microarray and conventional molecular techniques the variation of gene expression in lung from rabbits treated with slow-rate saline infusions. This analysis indicates that even a condition characterized by a small increase in extravascular water can have a significant influence on the inflammatory milieu. In this regard, cytokines, in particular TNFα, can be considered early mediators capable of inducing secondary effects on the injured tissue. Moreover, two MT1 genes were strongly up-regulated, data consistent with their role as protective molecules.

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.

Emerging evidence has demonstrated that miRNA sequences can regulate skeletal myogenesis by controlling the process of myoblast proliferation and differentiation. However, at present a deep analysis of miRNA expression in control and FSHD myoblasts during differentiation has not yet been derived. To close this gap, we used a next-generation sequencing (NGS) approach applied to in vitro myogenesis. Furthermore, to minimize sample genetic heterogeneity and muscle-type specific patterns of gene expression, miRNA profiling from NGS data was filtered with FC ≥ 4 (log(2)FC ≥ 2) and p-value

High-resolution cytogenetic analysis of a large number of women with premature ovarian failure (POF) identified six patients carrying different Xq chromosome rearrangements. The patients (one familial and five sporadic cases) were negative for Turner's stigmata and experienced a variable onset of menopause. Microsatellite analysis and fluorescent in situ hybridization (FISH) were used to define the origin and precise extension of the Xq anomalies. All of the patients had a Xq chromosome deletion as the common chromosomal abnormality, which was the only event in three cases and was associated with partial Xp or 9p trisomies in the remaining three. Two of the Xq chromosome deletions were terminal with breakpoints at Xq26.2 and Xq21.2, and one interstitial with breakpoints at Xq23 and Xq28. In all three cases, the del(X)s retained Xp and Xq specific telomeric sequences. One patient carries a psu dic(X) with the deletion at Xq22.2 or Xq22.3; the other two [carrying (X;X) and (X;9) unbalanced translocations, respectively] showed terminal deletions with the breakpoint at Xq22 within the DIAPH2 gene. Furthermore, the rearranged X chromosomes were almost totally inactivated, and the extent of the Xq deletions did not correlate with the timing of POF. In agreement with previous results, these findings suggest that the deletion of a restricted Xq region may be responsible for the POF phenotype. Our analysis indicates that this region extends from approximately Xq26.2 (between markers DXS8074 and HIGMI) to Xq28 (between markers DXS 1113 and ALD) and covers approximately 22 Mb of DNA. These data may provide a starting point for the identification of the gene(s) responsible for ovarian development and folliculogenesis.

We have isolated and characterised one PAC clone (dJ233C1) containing a linkage between alphoid and non-alphoid DNA. The non-alphoid DNA was found to map at the pericentromeric region of chromosome 20, both on p and q sides, and to contain homologies with one contig (ctg176, Sanger Centre), also located in the same chromosome region. At variance with the chromosome specificity shown by the majority of non-alphoid DNA, a subset of alphoid repeats derived from the PAC yielded FISH hybridisation signals located at the centromeric region of several human chromosomes, belonging to three different suprachromosomal families. The evolutionary conservation of this boundary region was investigated by comparative FISH experiments on chromosomes from great apes. The non-alphoid DNA was found to have undergone events of expansion and transposition to different pericentromeric regions of great apes chromosomes. Alphoid sequences revealed a very wide distribution of FISH signals in the great apes. The pattern was substantially discordant with the data available in the literature, which is essentially derived from the central alphoid subset. These results add further support to the emerging opinion that the pericentromeric regions are high plastics, and that the alpha satellite junctions do not share the evolutionary history with the main subsets.

The aim of the present study was to evaluate the effect of iron overload on gene expression in HepG2 cells by differential display. Iron-treated cells showed a 50% decrease in apolipoprotein B100 (Apo B100) and a 2- and 3-fold increase in semaphorin cd100 and aldose reductase mRNA, respectively, with parallel variations in Apo B100 and aldose reductase proteins. These effects were time-dependent. Vitamin E prevented the increase in aldose reductase expression, but had no effect on Apo B100 and semaphorin cd100. Treatment with hydrogen peroxide and 4-hydroxy-2,3-nonenal increased only aldose reductase mRNA. These data suggest that iron can affect mRNA levels by lipid peroxidation-dependent and -independent pathways.

Nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) modulate iron regulatory protein (IRP) activity and may, therefore, affect iron uptake through transferrin receptor expression. However, iron also enters the cell as nontransferrin-bound iron (NTBI), and the aim of this study was to evaluate the effects of NO donors on NTBI transport in HepG2 cells, a model of liver physiology. Incubation with SNP and SNAP led to a time- and concentration-dependent reduction in Fe3+ and Fe2+ uptake, thus indicating an effect on the transporter rather than on the reductase. In terms of Fe2+ uptake, no variations in the Michaelis-Menten constant (Km) and a reduction in maximum uptake (Vmax) (50, 33, and 16.6 fmol/microgram protein/min in control, SNP-, and SNAP-treated cells, respectively) were detected, which suggested a decrease in the number of putative NTBI transport protein(s). Gel shift assays showed that IRP activity was reduced by SNP and slightly increased by SNAP. Northern blot analysis of transferrin receptor messenger RNA (mRNA) levels showed variations similar to those observed for IRPs, but both NO donors increased L-ferritin mRNA levels and had no effect on the stimulator of Fe transport (SFT) mRNA. In conclusion, NO donors significantly reduce NTBI transport in HepG2 cells, an effect that seems to be IRP and SFT independent. Moreover, the reduction in NTBI uptake after NO treatment suggests that this form of iron may play a minor role in the increased hepatic iron stores observed in inflammation or that other liver cells are more involved in this pathological condition.

Southern-blot hybridization and partial sequencing of the pol and env genes were used to characterize BLV-integrated provirus of seropositive cattle from two dairy herds in northern Italy. Comparison of the data obtained with those of previously characterized BLV strains from other geographic areas (Australia, Belgium, Japan and USA) revealed the presence of a viral variant (BLV-12), which showed both conserved and unique features. Regarding the gp51 envelope glycoprotein, the BLV-12 variant showed: 1. A high extent of conservation, which included potential glycosylation sites and cysteine residues; 2. Three unique amino acid residues not present in any of the other BLV strains analysed; and 3. Some variability at the level of one (G) of the three (F, G and H) conformational epitopes, which is probably important in the process of infection. These results agree with the suggestion that the sequence variability of the gp51 glycoprotein preferentially involves structures whose change is thought to underlie the phenomenon of escape from immune surveillance.

Premature ovarian failure (POF) is a heterogeneous disorder whose aetiology is still unknown. Recently, the autosomal FOXL2 gene, highly expressed in the adult ovary, has been correlated with the disorder. FOXL2 mutations, causing a truncation of the FOXL2 protein in the forkhead domain or in the poly-Ala tract lead to blepharophimosis –ptosis–epicanthus –inversus syndrome associated with POF (BPES I). Interestingly, in two out of 70 idiopathic POF patients, a 30 bp deletion (898 –927del) and a missense mutation (1009T ! A) were identified. To further evaluate the correlation between POF and FOXL2 mutations, 120 phenotypically normal women affected by POF were analysed by direct sequencing of the FOXL2 coding region. The analysis did not reveal any mutation in the 240 analysed chromosomes, indicating that mutations in the FOXL2 coding region are rarely associated with non-syndromic POF.

We performed a detailed genomic investigation of the chimpanzee locus syntenic to human chromosome 4q35.2, associated to the facioscapulohumeral dystrophy. Two contigs of approximately 150 kb and 200 kb were derived from PTR chromosomes 4q35 and 3p12, respectively: both regions showed a very similar sequence organization, including D4Z4 and Beta satellite linked clusters. Starting from these findings, we derived a hypothetical evolutionary history of human 4q35, 10q26 and 3p12 chromosome regions focusing on the D4Z4–Beta satellite linked organization. The D4Z4 unit showed an open reading frame (DUX4) at both PTR 4q35 and 3p12 regions; furthermore some subregions of the Beta satellite unit showed a high degree of conservation between chimpanzee and humans. In conclusion, this paper provides evidence that at the 4q subtelomere the linkage between D4Z4 and Beta satellite arrays is a feature that appeared late during evolution and is conserved between chimpanzee and humans., Highlights ► A detailed genomic analysis of the PTR locus syntenic to human chromosome 4q35.2. ► PTR 4q35 and 3p12 regions carried a very similar D4Z4 and Beta satellite linked clusters. ► We derived a presumable evolutionary history of human 4q35, 10q26 and 3p12 regions. ► PTR and HSA subregions of the Beta satellite showed a high degree of conservation. ► 4q D4Z4–Beta satellite linked arrays appeared very late during evolution.

Background: Determine global gene dysregulation affecting 4q-linked (FSHD-1) and non 4q-linked (FSHD-2) cells during early stages of myogenic differentiation. This approach has been never applied to FSHD pathogenesis. Methodology/Principal Findings: By in vitro differentiation of FSHD-1 and FSHD-2 myoblasts and gene chip analysis we derived that gene expression profile is altered only in FSHD-1 myoblasts and FSHD-2 myotubes. The changes seen in FSHD-1 regarded a general defect in cell cycle progression, probably due to the upregulation of myogenic markers PAX3 and MYOD1, and a deficit of factors (SUV39H1 and HMGB2) involved in D4Z4 chromatin conformation. On the other hand, FSHD-2 mytubes were characterized by a general defect in RNA metabolism, protein synthesis and degradation and, to a lesser extent, in cell cycle. Common dysregulations regarded genes involved in response to oxidative stress and in sterol biosynthetic process. Interestingly, our results also suggest that miRNAs might be implied in both FSHD-1 and FSHD-2 gene dysregulation. Finally, in both cell differentiation systems, we did not observe a gradient of altered gene expression throughout the 4q35 chromosome. Conclusions/Significance: FSHD-1 and FSHD-2 cells showed, in different steps of myogenic differentiation, a global deregulation of gene expression rather than an alteration of expression of 4q35 specific genes. In general, FSHD-1 and FSHD-2 global gene deregulation interested common and distinctive biological processes. In this regard, defects of cell cycle progression (FSHD-1 and to a lesser extent FSHD-2), protein synthesis and degradation (FSHD-2), response to oxidative stress (FSHD-1 and FSHD-2), and cholesterol homeostasis (FSHD-1 and FSHD-2) may in general impair a correct myogenesis. Taken together our results recapitulate previously reported defects of FSHD-1, and add new insights into the gene deregulation characterizing both FSHD-1 and FSHD-2, in which miRNAs may play a role. © 2011 Cheli et al.

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with beta 2-microglobulin (beta 2m) by neuroblastoma (NB) cell lines IMR-32 and LA-N-1. The cells, which express barely detectable amounts of beta 2m-free (L31-positive molecules) and beta 2m-complexed HLA class I antigens (W6.32- and BBM.1-reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon-gamma (rIFN-gamma) treatment led IMR-32 and LA-N-1 cells to almost exclusively express beta 2m-complexes HLA class I heavy chains. Surface beta 2m-free MHC class I molecules displayed a molecular mass of approximately 45 kDa and did not bind exogenously added beta 2m. No changes in the synthesis of either HLA class I and beta 2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post-translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of beta 2m-free class I heavy chains is a feature of other cell types, such as NB cells.

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.

A variety of chromatin remodeling complexes are thought to orchestrate transcriptional programs that lead neuronal precursors from earliest commitment to terminal differentiation. Here we show that mammalian neurons have a specialized chromatin remodeling enzyme arising from a neurospecific splice variant of LSD1/KDM1, histone lysine specific demethylase 1, whose demethylase activity on Lys4 of histone H3 has been related to gene repression. We found that alternative splicing of LSD1 transcript generates four full-length isoforms from combinatorial retention of two identified exons: the 4 aa exon E8a is internal to the amine oxidase domain, and its inclusion is restricted to the nervous system. Remarkably, the expression of LSD1 splice variants is dynamically regulated throughout cortical development, particularly during perinatal stages, with a progressive increase of LSD1 neurospecific isoforms over the ubiquitous ones. Notably, the same LSD1 splice dynamics can be fairly recapitulated in cultured cortical neurons. Functionally, LSD1 isoforms displayin vitroa comparable demethylase activity, yet the inclusion of the sole exon E8a reduces LSD1 repressor activity on a reporter gene. Additional distinction among isoforms is supported by the knockdown of neurospecific variants in cortical neurons resulting in the inhibition of neurite maturation, whereas overexpression of the same variants enhances it. Instead, perturbation of LSD1 isoforms that are devoid of the neurospecific exon elicits no morphogenic effect. Collectively, results demonstrate that the arousal of neuronal LSD1 isoforms pacemakes early neurite morphogenesis, conferring a neurospecific function to LSD1 epigenetic activity.

Background Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects. Results In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation. Conclusion We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.

Murine monoclonal antibodies (mAbs) M38 and L31 define two epitopes of a surface protein of activated lymphocytes and monocytes. It has been shown that M38 also defines a crossreactive epitope of human immunodeficiency virus type 1 (HIV-1) gp120 (Beretta et al., 1987. Eur. J. Immunol. 17: 1793). The mAb inhibits syncytia formation driven by HIV-1-infected cells. The surface protein was demonstrated to be a class I MHC alpha chain, by sequence analysis of the corresponding cDNA and by immunological means. The epitopes defined by mAbs M38 and L31 are monomorphic and hidden (i.e., inaccessible to antibodies) on native HLA molecules expressed by resting cells, but can be evidenced on denatured proteins by Western blot analysis. The two epitopes become accessible after activation processes have been implemented, likely reflecting a conformational alteration of alpha chains (such as that described by Schnabl et al. 1990. J. Exp. Med. 171:1431). Consistent with molecular data are the results of functional analysis, which indicate that the molecule recognized by M38 and L31 is a gate for pleiotropic negative signals, since the two mAbs were shown to inhibit monocyte antigen presentation and lymphocyte mitogenic proliferation, respectively.

Background In order to obtain insights into the functionality of the human 4q35.2 domain harbouring the facioscapulohumeral muscular dystrophy (FSHD) locus, we investigated in African apes genomic and chromatin organisations, and the nuclear topology of orthologous regions. Results A basic block consisting of short D4Z4 arrays (10–15 repeats), 4q35.2 specific sequences, and approximately 35 kb of interspersed repeats from different LINE subfamilies was repeated at least twice in the gorilla 4qter. This genomic organisation has undergone evolutionary remodelling, leading to the single representation of both the D4Z4 array and LINE block in chimpanzee, and the loss of the LINE block in humans. The genomic remodelling has had an impact on 4qter chromatin organisation, but not its interphase nuclear topology. In comparison with humans, African apes show very low or undetectable levels of FRG1 and FRG2 histone 4 acetylation and gene transcription, although histone deacetylase inhibition restores gene transcription to levels comparable with those of human cells, thus indicating that the 4qter region is capable of acquiring a more open chromatin structure. Conversely, as in humans, the 4qter region in African apes has a very peripheral nuclear localisation. Conclusion The 4q subtelomere has undergone substantial genomic changes during evolution that have had an impact on chromatin condensation and the region's transcriptional regulation. Consequently, the 4qter genes in African apes and humans seem to be subjected to a different strategy of regulation in which LINE and D4Z4 sequences may play a pivotal role. However, the effect of peripheral nuclear anchoring of 4qter on these regulation mechanisms is still unclear. The observed differences in the regulation of 4qter gene expression between African apes and humans suggest that the human 4q35.2 locus has acquired a novel functional relevance.

BACKGROUND: Studies attempting to precisely define the range of fragile mental retardation 1 (FMR1) expansions and its inf luence in premature ovarian failure (POF) manifestation are partially lacking. To this aim, we evaluated a large cohort of POF patients for the size and, in selected cases, for the sequence of the CGG expansion. Furthermore, the correlation between POF and X-inactivation was investigated in FRAXA families. METHODS: By fluorescent PCR, 190 POF and 200 control women were sized for the CGG tract; some subjects were also characterized by sequencing and for the FMR1 activation ratio. RESULTS AND CONCLUSION: We found a significant association (19/190, 10%, P < 1 x 10(-6)) between POF and FMR1 premutation (range 63-163 repeats) and a significant enrichment (9/190, 4.7%, P = 0.021) of POF carriers of intermediate expansions (range 41-58 repeats). Interestingly, intermediate alleles were entirely composed of CGG repeats. Furthermore, the analysis of three pairs of siblings with similar FMR1 expansions and discordant for the POF phenotype showed a direct correlation between the expression of the intermediate/premutated allele and POF manifestation. The results obtained strengthen the correlation between FMR1 expansion and POF and suggest that the manifestation of the ovarian dysfunction could be influenced both by the pattern of interruption of the CGG repeat and by X-inactivation.

In this article, we report studies on the evolutionary history of beta satellite repeats (BSR) in primates. In the orangutan genome, the bulk of BSR sequences was found organized as very short stretches of approximately 100 to 170 bp, embedded in a 60-kb to 80-kb duplicated DNA segment. The estimated copy number of the duplicon that carries BSR sequences ranges from 70 to 100 per orangutan haploid genome. In both macaque and gibbon, the duplicon mapped to a single chromosomal region at the boundary of the rDNA on the marker chromosome (chromosome 13 and 12, respectively). However, only in the gibbon, the duplicon comprised 100 bp of beta satellite. Thus, the ancestral copy of the duplicon appeared in Old World monkeys ( approximately 25 to approximately 35 MYA), whereas the prototype of beta satellite repeats took place in a gibbon ancestor, after apes/Old World monkeys divergence ( approximately 25 MYA). Subsequently, a burst in spreading of the duplicon that carries the beta satellite was observed in the orangutan, after lesser apes divergence from the great apes-humans lineage ( approximately 18 MYA). The analysis of the orangutan genome also indicated the existence of two variants of the duplication that differ for the length (100 or 170 bp) of beta satellite repeats. The latter organization was probably generated by nonhomologous recombination between two 100-bp repeated regions, and it likely led to the duplication of the single Sau3A site present in the 100-bp variant, which generated the prototype of Sau3A 68-bp beta satellite tandem organization. The two variants of the duplication, although with a different ratios, characterize the hominoid genomes from the orangutan to humans, preferentially involving acrocentric chromosomes. At variance to alpha satellite, which appeared before the divergence of New World and Old World monkeys, the beta satellite evolutionary history began in apes ancestor, where we have first documented a low-copy, nonduplicated BSR sequence. The first step of BSR amplification and spreading occurred, most likely, because the BSR was part of a large duplicon, which underwent a burst dispersal in great apes' ancestor after the lesser apes' branching. Then, after orangutan divergence, BSR acquired the clustered structural organization typical of satellite DNA.

Background: Balanced X;autosome translocations interrupting the 'critical region' of the long arm of the human X chromosome are often associated with premature ovarian failure (POF). However, the mechanisms leading to X-linked ovarian dysfunction are largely unknown, as the majority of the X chromosome breakpoints have been mapped to gene-free genomic regions. A few genes have been found to be interrupted, but their role has never been clarified. Methods and Results: By fine mapping of the X chromosome breakpoint of an X;autosome balanced translocation, we identified a new interrupted gene, POF1B. We performed a mutation analysis of POF1B and of another gene previously identified, DACH2, localized similar to700 kb distal in Xq21, in a cohort of >200 Italian POF patients. Rare mutations were found in patients in both genes. Conclusions: Our findings could not demonstrate any involvement of POF1B, but suggest that rare mutations in the DACH2 gene may have a role in the POF phenotype.

A cDNA clone up-regulated in hydraulic lung edema in rabbit showed high similarity with human RDH10 mRNA, which encodes a protein involved in retinoic acid metabolism. We defined the organization of the human gene, which includes a unique transcriptional start site, a coding region with six translated exons and a 3′ untranslated region containing at least two used polyadenylation sites. The two poly(A) signals are responsible for the production of the 3 and 4 kb RDH10 mRNA isoforms detected in several human tissues and cell lines.

BACKGROUND: Premature ovarian failure (POF) is a secondary hypergonadotrophic amenorrhoea affecting 1–3% of females, whose aetiology is almost unknown. However, inhibin alpha gene (INHα) has recently been indicated as candidate in POF pathogenesis. METHODS: We analysed patients affected by POF (n 157) for the missense mutation (769G→A transition) in the exon 2 of the INHα gene. The same analysis was carried out on early menopause (EM) (n 36) and primary amenorrhoea (n 12) patients. RESULTS: The incidence of the mutation was significantly more frequent within both POF (7/157, 4.5%) (Fisher’s exact test, P 0.030) and primary amenorrhoea (3/12, 25%) (Fisher’s exact test, P < 0.001) patients, compared with the control population of women (0/100), who experienced physiological menopause. No mutation was found in EM patients. Furthermore, the likelihood of finding the mutation was statistically significant in familial (5/65; 7.7%) (Fisher’s exact test, P < 0.01) but not in sporadic (2/92; 2.2%) (Fisher’s exact test, P not significant) POF, compared with the control group. The analysis of pedigrees showing the inheritance of the 769G→A mutation and POF strengthens the concept of the disease heterogeneity, since the POF phenotype was not always associated with the mutation. Moreover, a higher prevalence of the C allele of a single nucleotide polymorphism (129C→T), located in the 5-UTR of the INHα gene, was observed in POF patients (80.3%) than in the control group (66.7%) (Fisher’s exact test, P 0.014). CONCLUSION: These data strengthen the concept of the INHα gene as a candidate for ovarian failure.

We report the molecular analysis of a 130-kb DNA region containing a junction between beta and non-beta satellite DNA from chromosome 15p. The genomic region is characterized by beta satellite blocks intermingled with variants of the D4Z4 repeat, and duplicons from 4q24 and 4q35. Besides the p-arm of acrocentric chromosomes, the duplicons showed a wide genomespread involving pericentromeric, sub-telomeric, and interstitial regions. In this regard, the paralogous sequences were characterized by a high similarity index (96%), thus indicating a recent transposition during the evolution. The acrocentrics differedwith regard to the location of the 4q24 paralogous region, since it mapped on the p-arm of chromosomes 13–15 and 21, but only on 22q11.2. Conversely, the 4q35 duplication marked the p-arm of all the acrocentrics. In different individuals, the short arm of acrocentric chromosomes revealed a great variability of sequence representation and location at p11 and/or p13 for both the 4q24 and 4q35 duplications. The studied genomic region from chromosome 15p, of which a contig of approximately 200 kb has been derived, could lead to more detailed investigations into the sequence organization and possible biological function of chromosome regions that are located close to the rDNA array.

Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25\% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.

Clone CSA7 is a CHEF18 hamster cell line that shows an increased intracellular accumulation of dCTP. To localize the mutations that accumulate spontaneously in a functional gene of such a mutator phenotype, independent CSA7 mutants of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene were isolated and screened by a polymerase chain reaction-single strand conformation polymorphism technique. Sixty-two percent of mutants produced detectable changes of the strand migration profile and the mutations were preferentially localized in the exons 3 (31%) and 6 (62%). The sequencing of such exons revealed that the rate of C base incorporation was the major mutation pathway and that the A base of a GGA sequence was the preferential site of misincorporation.

Transgenic mice were produced in which human HLA-Cw4 is stably integrated, behaves as a single Mendelian trait, and, being under the transcriptional control of human CD2, is selectively and efficiently expressed in T lymphocytes. These mice were used as a model system to determine whether HLA-type C molecules can be exposed on the surface of activated lymphocytes as free heavy chains, non-associated with beta2-microglobulin (beta2m). In our transgenic mice we could identify HLA-Cw4 molecules either as free chains or as beta2m-associated molecules by the use of monoclonal antibodies specific for either conformation of HLA class I and nonreactive to mouse H2 molecules. Resting mouse lymphocytes were shown by western transfer analysis to contain sizeable amounts of HLA-Cw4 free chains, but they exposed on their surface HLA-Cw4 only in association with beta2m, as indicated by flow cytometric measurements. Conversely, where the content of total HLA-Cw4 was increased, lectin-activated mouse lymphocytes exposed on their outer cell membrane HLA-Cw4 molecules in both conformations, namely, also as free heavy chains. Isoelectrofocusing analysis confirmed the presence of both HLA-Cw4 molecular conformations in activated T cells and indicated that HLA-Cw4 heavy chains can bind to mouse beta2m with the same low affinity displayed for human beta2m. The results of our experiments led us to conclude that (1) association with beta2m is not necessary for the exposure of HLA-C on the surface of activated T lymphocytes and (2) cell activation affects the balance between the two conformational forms of HLA-C.

Classical serological methods and Southern blot hybridization for the diagnosis of bovine leukaemia virus (BLV) infection have been compared during the first nine months of life of offspring from BLV serum-negative and serum-positive dams belonging to a Friesian dairy herd in Italy. At birth, 9/13 calves analysed showed serum positivity for anti-gp60 BLV antibodies by agar immunodiffusion and/or by ELISA. However, only two calves were positive for BLV integrated proviruses in their lymphocyte DNA. At six months of age, anti-gp60 BLV antibodies and proviral DNA positivities were simultaneously shown only by the two cattle identified as DNA-positive at birth. This pattern remained constant up to nine months of age. Furthermore, analysis of the molecular characteristics of BLV integrated proviruses, carried out by using, as probes, the almost complete proviral genome (Belgian isolate) or a subclone of the env gene radioactively labelled or chemically modified, revealed that the calves under study were infected by a different isolate (Japanese isolate) and that, in one of the cattle, the majority of integrated proviruses was characterized by deletions probably located in the 5′ half of the proviral genome.

A serological cross-reactivity between env gp120 glycoprotein of the human immunodeficiency virus (HIV) and a human cellular surface protein has been defined by a monoclonal antibody (M38) raised against HIV. The cellular antigen is a protein of ca. 80 kDa expressed on a small fraction of mononuclear cells in peripheral blood and in lymph nodes. The protein behaves as an activation antigen of the monocytic lineage since it is expressed by monocytes in plastic-adherent culture conditions and by interferon-gamma-treated monocytes and pro-monocytic U937 cells. The protein is involved in antigen presentation since the antibody efficiently inhibits the proliferation of responsive lymphocytes in autologous tetanus toxoid presentation assays. In the T lymphoblastoid line H9, the protein is present in very small amounts, is not induced by interferon-gamma and increases after HIV infection. Sera from lymphoadenopathy syndrome and acquired immunodeficiency syndrome (AIDS) patients fail to detect the cellular protein, although containing antibodies reacting with gp120. We propose that both viral and cellular structures recognized by the monoclonal antibody (mAb) are involved in interactions with CD4 molecules of T helper lymphocytes and that such molecular mimicry might be relevant in the pathology of HIV infection. This view is supported by the finding that BL/10T4, a CD4-specific mAb, binds to M38 neutralizing its interactions with HIV and with monocytes. mAb M38 thus behaves as the internal image of CD4. This single property would explain all its diverse binding characteristics.

By means of restriction enzymes analysis and molecular hybridization, the distribution of repeated DNA families has been studied in the different DNA components into which the human genome can be fractionated by density gradient techniques. Three classes of DNA molecules have been analyzed: i) an homogeneous DNA component (satellite-like sequences; ϱ=1.696g/cm3, 3% of total DNA, AT repeated ), ii) AT rich (ϱ=1.698g/cm3, 30% of total DNA, AT main-band ) and GC rich (ϱ=1.708 g/cm3, 6% of total DNA, GC main-band ) DNA components. By this approach we have observed that Sau3A digestion of GC main-band gives rise to two bands of 75bp and 150bp, absent or under-represented in both AT rich DNA components. A preliminary characterization of these DNA fragments suggests that they contain one or more families of repeated sequences which fail to hybridize to EcoRI, HindIII and AluI families of repeats. In addition, we have observed that EcoRI sequences (α-RI DNA) are under-represented in GC main-band and show the same clustered organization in both AT rich DNA components.

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.

Summary As little as 10 pg of BHV-I DNA is detected by dot-blot hybridization with 32P-labelled DNA from plasmid pHV-2, a recombinant bacterial plasmid containing 12·5 kb of the BHV-I genome. The probe shows very low homology with other herpetic viruses and no hybridization with bovine genomic DNA. An assay system has been developed to detect viral particles in the presence of bovine semen in reconstituted mixtures. By using this procedure the presence of BHV-I DNA has been demonstrated in the semen sample from a serum-positive animal found to be negative by the cell culture inoculation technique.

A bisexual species of the genus Artemia (Crustacea, Phyllopoda), Artemia franciscana Barigozzi of San Francisco Bay and a parthenogenetic population of Artemia sp. of Tsing-Tao (China), both with 42 chromosomes, were compared with respect to the microscopic structure of the interphase larval nucleus, the microscopical structure of the prophase chromosomes and the DNA structure. — Artemia franciscana exhibits several chromocenters in the resting nucleus, heterochromatic blocks located at the end of the prophase chromosomes, and a large amount of repetitive DNA (Alu I 110-bp fragments). The other Artemia sp. lacks chromocenters, heterochromatic blocks in the chromosomes, and the Alu I DNA. The two populations thus differ by a remarkable amount of repetitive DNA.

The genus Artemia (Crustacea, Phyllopoda) is widely distributed all over the world as a result partly of natural colonization and partly of spread by birds and man. Artemia offers a very interesting model for speciation studies, since the genus comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, heteroploidy and polyploidy). The finding of the clustered repetitive AluI DNA family in the heterochromatin of A. franciscana can provide a useful tool for investigating the relationship between the members of the genus Artemia at the molecular level. Sixteen strains of Artemia, comprising sibling species and parthenogenetic populations, were analysed for the presence of AluI repetitive DNA by dot-blot hybridization. The observed variation in the content of repetitive DNA together with genetical, biological and geological data, support the hypothesis that Artemia living in the New World are derived from ancestral species that evolved in the Mediterranean area.

Sau3A digestion of human G + C-rich DNA molecules yields discrete bands of approximately 70 and 140 base-pairs, under-represented in A + T-rich DNA molecules and in total DNA. We have cloned the 70 base-pair band in a plasmid vector and isolated a representative recombinant clone that identifies a new human family of repeats, the Sau3A family. The new family has been characterized for a number of parameters: genomic organization; reiteration frequency; sequence analysis; and distribution in a human genomic library. The Sau3A sequence (68 base-pairs in length, 53% G + C) is present in approximately 4 X 10(4) copies/haploid genome; the family is characterized by a cluster organization and is confined to a limited fraction (0.5%) of phages of a human genomic library. Southern blot hybridizations of the cloned sequence to restriction digests of total human DNA and of isolated genomic clones does not show the involvement of Sau3A blocks in long-range periodicities for any of the enzymes tested. The data suggest either a high sequence variability in the family or a complex organization of Sau3A sequence domains.

The DNA extracted from human leukemic leucocytes has been fractionated on a methylated albumin kieselguhr (MAK) column. The different fractions obtained have been reannealed to a Cot value of 20 mol times sec/1 to study the distribution of the intermediate DNA on the MAK column. Intermediate DNA contains two components, one (CsCl density after reannealing, 1.703 g/ml) obtained by reannealing high molecular weight DNA, the other (CsCl density after reannealing, 1.707 g/ml) obtained only by reannealing sonicated low molecular weight DNA. High molecular weight intermediate DNA (1.703 component) is eluted early from the MAK column in the fractions corresponding to the main DNA peak, while low molecular weight intermediate DNA (1.707 component) is more widespread on the MAK column, but appears to be enriched in the fractions eluted later. The possibility is discussed that the latter component is interspersed in that part of the genome which is apparently more homogeneous in density in an analytical CsCl gradient, and is absent on the skewed, more heterogeneous, heavy side of the main DNA in CsCl.

The antibiotic aphidicolin inhibited in vitro deoxyribonucleic acid synthesis catalyzed by crude yeast extracts and by partially purified yeast deoxyribonucleic acid polymerases. The mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerase I was noncompetitive with deoxyguanosine 5'-triphosphate, deoxyadenosine 5'-triphosphate, and deoxythymidine 5'-triphosphate and was of the mixed type with deoxycytidine 5'-triphosphate. The relative ratio of enzyme to the template-initiator complex was important for detecting the inhibitory effect of the antibiotic. The inhibition of in vitro deoxyribonucleic acid synthesis by aphidicolin was reversible, and the effect on yeast deoxyribonucleic acid polymerase might have been partially mediated by some supplementary factor(s).

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with Xba I and Hae III (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [ 3 H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing Eco RI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [ 3 H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with Xba I and Hae III, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the Xba I 2.4 kb fragment becomes undetectable in late S.

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp)) were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a “new” family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.

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 .

The DNA extracted in macromolecular form from human leukemic leucocytes has been fractionated in Ag+- Cs2SO4 and Hg++-Cs2SO4 preparati

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.

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.

The Sau3A family is a human, clustered, highly repetitive, GC-rich DNA family. In situ hybridization studies with a plasmid carrying a Sau3A monomer as a probe have shown that Sau3A sequences are preferentially concentrated in the heterochromatic regions of human acrocentric chromosomes (D and G groups, both in pericentromeric regions and in cytological satellites) and in pericentromeric heterochromatin of chromosome 1. The same chromosomal locations were observed by using as probes two recombinant phages which carry Sau3A-positive genomic sectors. The two sectors differ for the relative proportions of monomer and multiples of Sau3A repeats, which show different extents of homology to the cloned monomer, and for the presence, in one of the two, of a small amount of an unrelated repeat (alphoid DNA). The similarity of the results obtained with the three probes suggests that heterogeneous Sau3A repeats share the same chromosomal localizations and that the two analyzed genomic sectors may not contain significant amounts of repetitive DNAs other than the Sau3A family. A comparison between the chromosomal locations of Sau3A and EcoRI families of repeats has confirmed that each family is characterized by specific chromosomal locations and that single heterochromatic regions may contain both.

Moloney murine leukemia virus c-DNA hybridizes mainly with cellular middle repeated sequences of NIH-Swiss mouse spleen DNA, fragmented to different lengths, denatured and renatured to an intermediate value of Cot, and fractionated in an Ag+-Cs2SO4 preparative gradient suitable to separate unique, middle repeated and highly repeated DNA.