Relating centromeric topography in fixed human chromosomes to α-satellite DNA and CENP-B distribution.

Despite extensive analyses on the centromere and its associated proteins, detailed studies of centromeric DNA structure have provided limited information about its topography in condensed chromatin. We have developed a method with correlative fluorescence light microscopy and atomic force microscopy that investigates the physical and structural organization of α-satellite DNA sequences in the context of its associated protein, CENP-B, on human metaphase chromosome topography. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicates that CENP-B and α-satellite DNA are distributed distinctly from one another and relative to observed centromeric ridge topography. Our approach facilitates correlated studies of multiple chromatin components comprising higher-order structures of human metaphase chromosomes.

Allele specificity of DNA replication timing in the Angelman/Prader-Willi syndrome imprinted chromosomal region.

DNA replication within chromosome 15q11-q13, a region subject to genomic imprinting, was examined by fluorescence in situ hybridization. Asynchronous replication between homologues was observed in cells from normal individuals and in Prader-Willi (PWS) and Angelman syndrome (AS) patients with chromosome 15 deletions but not in PWS patients with maternal uniparental disomy. Opposite patterns of allele-specific replication timing between homologous loci were observed; paternal early/maternal late at D15S63, D15S10 and the gamma-aminobutyric acid receptor beta 3 subunit gene (GABRB3); and maternal early/paternal late at the more distal gamma-aminobutyric acid receptor alpha 5 subunit gene (GABRA5). At the most distal locus examined, D15S12, both patterns of allele-specific replication timing were detected.

Maternal but not paternal transmission of 15q11-13-linked nondeletion Angelman syndrome leads to phenotypic expression.

Angelman syndrome (AS) may result from either maternally inherited deletions of chromosome 15q11-13 or from paternal uniparental disomy for chromosome 15. This is in contrast to Prader-Willi syndrome (PWS), which is caused by either paternal deletion of this region or maternal disomy for chromosome 15. However, 40% of AS patients inherit an apparently intact copy of chromosome 15 from each parent. We now describe a family in which three sisters have given birth to four AS offspring who have no evidence of deletion or paternal disomy. We show that AS in this family is caused by a mutation in 15q11-13 that results in AS when transmitted from mother to child, but no phenotype when transmitted paternally. These results suggest that the loci responsible for AS and PWS, although closely linked, are distinct.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator.

Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.

Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.

Activation of the Lbc Rho exchange factor proto-oncogene by truncation of an extended C terminus that regulates transformation and targeting.

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted alpha-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the alpha-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

Angelman syndrome: validation of molecular cytogenetic analysis of chromosome 15q11-q13 for deletion detection.

In a series of 18 individuals comprising parents of Angelman syndrome (AS) patients and AS patients with large deletions, microdeletions, and no deletions, we utilized fluorescence in situ hybridization (FISH) with genomic phage clones for loci D15S63 and GABRB3 for deletion detection of chromosome 15q11-q13. Utilization of probes at these loci allows detection of common large deletions and permits discrimination of less common small deletions. In all individuals the molecular cytogenetic data were concordant with the DNA deletion analyses. FISH provides an accurate method of deletion detection for chromosome 15q11-q13.

Cytogenetic and molecular studies in the Prader-Willi and Angelman syndromes: an overview.

The majority of patients with Angelman syndrome and Prader-Willi syndrome have a cytogenetic and molecular deletion of chromosome 15q11q13 with the primary difference being in the parental origin of deletion. Our current understanding of the cytogenetics and molecular genetics of these 2 clinically distinct syndromes will be discussed in this review.

Supernumerary inv dup(15) in a patient with Angelman syndrome and a deletion of 15q11-q13.

We have studied a patient with Angelman syndrome (AS) and a 47,XY,+inv dup(15) (pter-->q11::q11-->pter) karyotype. Molecular cytogenetic studies demonstrated that one of the apparently normal 15s was deleted at loci D15S9, GABRB3, and D15S12. There were no additional copies of these loci on the inv dup(15). The inv dup(15) contained only the pericentromeric sequence D15Z1. Quantitative DNA analysis confirmed these findings and documented a standard large deletion of sequences from 15q11-q13, as usually seen in patients with AS. DNA methylation testing at D15S63 showed a deletion of the maternally derived chromosome 15q11-q13 on one of the apparently cytogenetically normal 15s, and not by the presence of an inv dup(15). This is the fourth patient with an inv dup(15) and AS or Prader Willi syndrome, who has been studied at the molecular level. In all cases an additional alteration of chromosome 15 was identified, which was hypothesized to be the cause of the disease. Patients with inv dup(15)s may be at increased risk for other chromosome abnormalities involving 15q11-q13.