Isochores and replication time zones: a perfect match.

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties.

Analysis of an alternatively spliced exon of the neurofibromatosis type 1 gene in cultured melanocytes from patients with neurofibromatosis 1.

Neurofibromatosis type 1 (NF1) is characterized by clinical features that primarily affect tissues derived from the neural crest (neurofibromas, café-aulait macules). Because aberrant regulation of alternative splicing in the NF1 gene transcript may be of functional significance, cultured melanocytes from café-aulait macules (CALM), as an example of benign NF1 lesions, were examined for the expression of the different alternative splice products of this gene. Both kinds of NF1 messengers (type 1 and 2) were found not only in CALM melanocytes but also in keratinocytes, fibroblasts and blood cells. Except in blood cells, there was a predominance of the type 2 transcript. Melanocytes from NF1 patients and healthy donors showed similar expression patterns under several culture conditions. Our results suggest that the development of CALM does not correlate with a switch in the ratio of type 1 to type 2 NF1 messenger RNA.

Increased amount and contour length distribution of small polydisperse circular DNA (spcDNA) in Fanconi anemia.

Small polydisperse circular DNA (spcDNA) in Fanconi anemia (FA) was analyzed from cultured fibroblast-like cells by electron microscopy. Application of the mica-press adsorption technique for the semi-quantitative determination of spcDNA amounts to three FA and three normal control skin-derived fibroblast strains revealed 85-fold increased levels of spcDNA in the FA cells. An even higher excess over controls was suggested when the FA fibroblasts were propagated for up to 11 serial in vitro passages, consistent with the short replicative life-span of primary FA cells and their rapid transition into a poorly dividing state, in which spcDNA reportedly further increases. In addition, contour length distributions of gradient-purified spcDNA preparations from five FA fibroblast strains were compared with those from five normal control strains. Mean spcDNA contour lengths were significantly greater in the FA than in the control cells. The reported findings of increased spcDNA amounts and sizes in FA coincide with a similar association of chromosome instability and abnormal spcDNA formation previously observed in cultured cells derived from angiofibromas in tuberous sclerosis. Circumstantial evidence from the present study in the paradigmatic chromosome breakage syndrome FA further supports the suggestion that a common mechanism underlies chromosome instability and the surplus generation of spcDNA. Notably, this apparent mechanism is functional in homonuclear primary cell strains with a distinct inherited basis of their chromosome instability, and is not restricted to heteroploid and neoplastoid cell lines.

A comparison of the variability spectra of two genomic loci in a European group of individuals reveals fundamental differences pointing to selection or a population bottleneck.

Knowledge about the variability spectra of neutrally evolving sequences in a population is a prerequisite for the identification of genes, which may have been under positive selection during recent human evolution. Here, we report the results of a re-sequencing project of a presumably neutrally evolving chromosome 22 locus with a severely reduced recombination frequency in a group of 24 individuals of German origin. The comparison of these data with the results of a similar analysis of a chromosome 17 locus revealed striking differences, although the same group of individuals was used. For the chromosome 17 locus two well-separated groups of sequences, a positive value of Tajima's D and a TMRCA of 700,000 years were observed. In contrast, the sequences from the chromosome 22 locus were found to be relatively homogeneous, with no deep splits between subgroups; the obtained value for Tajima's D was negative and the TMRCA was only 260,000 years. These discrepancies may be explained by selection or demographic processes. Regarding demography, the most plausible explanation is the assumption of a severe bottleneck in the history of the European population: in the case of the chromosome 17 locus two ancient lineages passed this bottleneck; for the chromosome 22 locus it was only one ancient lineage.

A small deletion and an adjacent base exchange in a potential stem-loop region of the neurofibromatosis 1 gene.

A single-strand conformational polymorphism found in the DNA of a patient with neurofibromatosis 1 (NF1) was shown to be caused by a deletion of a CCACC or CACCT sequence and an adjacent transversion, located about 500 base pairs downstream from the region that codes for a functional domain of the NF1 gene product. This mutation could also be detected in the patient and in his affected daughter by heteroduplex analysis. The deletion removes the proximal half of a small potential stem-loop and interrupts the reading frame in exon 1. A severely truncated protein with a grossly altered carboxy terminus lacking one third of its sequence is expected to be formed from the mutant allele.

Single-cell PCR performed with neurofibroma Schwann cells reveals the presence of both alleles of the neurofibromatosis type 1 (NF1) gene.

It is commonly held that Schwann cells (SC) are the progenitor cells of benign neurofibromas. To test for loss of heterozygosity (LOH) at the neurofibromatosis 1 (NF1) gene locus, three intragenic polymorphic markers were analyzed after polymerase chain reaction amplification, starting from 98 single SC isolated from primary cultures of neurofibromas, of five informative NF1 patients. The patterns obtained did not provide evidence for LOH at the NF1 gene. LOH by nondisjunction, large deletions, or somatic recombination in SC seems not to be the mechanism of generation of neurofibromas.

A palindromic structure in the pericentromeric region of various human chromosomes.

The primate-specific multisequence family chAB4 is represented with approximately 40 copies within the haploid human genome. Former analyis revealed that unusually long repetition units ( > 35 kb) are distributed to at least eight different chromosomal loci. Remarkably varying copy-numbers within the genomes of closely related primate species as well as the existence of human specific subfamilies, which most probably arose by frequent sequence exchanges, demonstrate that chAB4 is an unstable genomic element, at least in an evolutionary sense. To analyze the chAB4 basic unit in more detail we established a cosmid contig and found it to be organized as inverted duplications of approximately 90 kb flanking a noninverted core sequence of approximately 60 kb. FISH as well as the analysis of chromosome-specific hybrid cell lines revealed a chromosomal localization of chAB4 on chromosomes 1, 3, 4, 9, Y, and the pericentromeric region of all acrocentrics. Furthermore, we can detect chAB4 sequences together with alpha satellites, beta satellites, and satellite III sequences within a single chromosome 22-specific YAC clone, indicating that chAB4 is located in close proximity to the centromere, at least on the acrocentrics. Hence, chAB4 represents an unstable genomic structure that is located just in the chromosomal region that is very often involved in translocation processes.

Analysis of human extrachromosomal DNA elements originating from different beta-satellite subfamilies.

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of beta-satellite DNA. Further experiments have shown that the three sequences belong to at least two different beta-satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that beta-satellite sequences obtained from the spcDNA of human cells have been assigned to beta-satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that beta-satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.

The size of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TSC) differs from that in fibroblasts.

Cell cultures were derived from angiofibromas of three patients with tuberous sclerosis (TSC), from the unaffected skin of these patients, and from the skin of five healthy donors. The length distributions of the small polydisperse circular DNA (spcDNA) fraction of these cell cultures were then analyzed. Nearly half the spcDNA molecules from the angiofibroma cultures were longer than 0.4 micron, whereas only about 7% exceeded this threshold in the spcDNA preparations from the skin fibroblast cultures. The percentage of the larger size class of spcDNA showed an increase at higher numbers of in vitro passages in all three types of cultures, but this effect was much more conspicuous in the angiofibroma-derived cultures than in those from the skin fibroblasts. An age-dependent increase in the overall amount of spcDNA was only seen in the angiofibroma-derived cultures. Our earlier finding of elevated amounts of spcDNA in angiofibroma cultures was confirmed in cultures from an additional TSC patient.

Evolution of the chAB4 multisequence family in primates.

Approximately 50 members of the primate-specific multisequence family chAB4 are located as clusters at eight different chromosomal loci within the human genome. The whole cloned region of chAB4 represents a single-copy or low-copy sequence in all nonhuman primates tested, with the exception of the chimpanzee, for which we found chAB4 copy numbers similar to those in the human. An Alu element was inserted into chAB4 after the divergence of the Old World monkeys from the hominoids but before chAB4 was amplified. The first amplification step could be dated after the great apes and the human diverged from the Old World monkeys. We have evidence that neither the copy numbers nor the chromosomal locations remained stable after this initial step and that gross alterations in the relative copy numbers of individual family members occurred even after the divergence of the human and the chimpanzee. Taken together, our data suggest that chAB4, in an evolutionary sense, is an unusually unstable sequence family.

Evidence for the presence of the second allele of the neurofibromatosis type 1 gene in melanocytes derived from café au laitmacules of NF1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited disorder caused by mutations in the NF1 gene on 17q11.2. Melanocytes cultured from cafe au lait macules (CALM) of patients with NF1 were analysed for loss of heterozygosity (LOH) at the NF1 locus using a 3'-flanking and four intragenic markers. None of the informative samples showed LOH. In addition, the X-inactivation pattern of melanocytes from CALM (n = 4) and from the unaffected skin of the patients (n = 3) suggests a monoclonal origin of the cells isolated from skin biopsies up to 2 cm2 in size.

Detection of a restriction site polymorphism within the human alpha-globin gene complex.

A mutant Rsa I restriction endonuclease site of high frequency has been identified in individuals of German, Greek, Italian, and Turkish origin. The mutation was found within the alpha-globin gene complex and is located 0.7 kb 5' to the alpha 2-globin gene. In individuals of central European origin 34 out of 58 chromosomes exhibited the alpha-gene linkage to the presence of the polymorphic site, and thus a preliminary estimate of the gene frequency for this allele would be 0.59. DNA analysis data of individuals derived from Mediterranean populations indicate a distribution of this polymorphic marker in similar frequencies.

Toward a survey of somatic mutation of the NF1 gene in benign neurofibromas of patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.

A deletion in the 5'-region of the neurofibromatosis type 1 (NF1) gene.

A new mutation, the first one close to the 5'-end of the neurofibromatosis type 1 (NF1) gene, was found when RNA preparations from various cell types of 15 NF1 patients were analysed by reverse transcription and subsequent multiplex polymerase chain reaction. This mutation removes the 84 bp of exon 3 precisely from the cDNA. Genomic Southern blots revealed a larger deletion with breakpoints within the introns flanking exon 3. This mutation suggests that the amino-terminal region of neurofibromin is functionally significant. When using this mutation to distinguish the wild type and mutant alleles, their expression could be analysed in neurofibroma fibroblasts, melanocytes from the unaffected skin, and those from a café-au-lait macule. In all these cell types, the products of both alleles were detected, confirming similar results obtained with a different NF1 gene mutation.

Mapping of members of the low-copy-number repetitive DNA sequence family chAB4 within the p arms of human acrocentric chromosomes: characterization of Robertsonian translocations.

Members of the long-range, low-copy-number repetitive DNA sequence family chAB4 are located on nine different human chromosome pairs and the Y chromosome, i.e. on the short arms of all the acrocentrics. To localize the chAB4 sequences more precisely on the acrocentrics, chAB4-specific probes together with rDNA and a number of satellite sequences were hybridized to metaphase chromosomes of normal probands and of carriers of Robertsonian translocations of the frequent types rob(13q14q) and rob(14q21q). The results demonstrate that chAB4 is located on both sides of the rDNA on all the acrocentrics; the exact location, however, may be chromosome specific. Chromosome 22, most probably, is the only chromosome where chAB4 is found in the direct neighbourhood of the centromere. Fluorescence in situ hybridization analyses of metaphase chromosomes of carriers of rob(21q22q) revealed breakpoint diversity for this rare type of Robertsonian translocation chromosome. A direct involvement of chAB4 sequences in recombination processes leading to the Robertsonian translocations analysed in this study can be excluded.

Characterization of an alphoid subfamily located near p-arm sequences on human chromosome 22.

The centromeric heterochromatin of all human chromosomes is composed of tandemly repeated alpha satellite DNA. Here we describe another alphoid subfamily that maps to human chromosome 22 as determined by FISH. The alphoid sequences were isolated from three YAC-clones carrying DNA from the pericentromeric region of the short arm of human chromosome 22 and limited amounts of alphoid DNA. This property enabled us to map the members of the subfamily to the border of the centromeric region and the short arm of the chromosome. The new alphoid subfamily may contribute to the closure of the gap remaining between the centromeric and short-arm maps of human chromosome 22.

On unequal allelic expression of the neurofibromin gene in neurofibromatosis type 1.

The autosomal dominantly inherited disease neurofibromatosis type 1 (NF1) is caused by mutations of a large gene comprising 59 exons, which code for a protein with 2818 amino acids called neurofibromin. Employing an expressed polymorphic site in exon 5 of the neurofibromin gene, the expression of its alleles was analysed quantitatively by scanning radioactive RT-PCR fragments of this exon prepared from the RNA of fibroblast cell cultures from 15 NF1 patients and of white blood cells from one NF1 patient. Thirteen of the RNA preparations yielded unequal amounts of the allelic messages. The deviations of the expression ratios (A2:A1) from 1.0 ranged from -0.9 to +25.8. The allelic messages were equally represented in the RNA preparations from five informative healthy donors. Apart from fibroblasts this phenomenon could also be detected in keratinocytes, melanocytes from normally pigmented skin and melanocytes from a café-au-lait spot of one patient. Only one of three patients affected by stop mutations exhibited unequal allelic expression. When nuclear RNA from 10 of the 13 patients was examined, equal amounts of the primary transcripts were found (average ratio A2/A1: 1.08 +/- 0.07 S.E.M.), indicating that unequal expression on the level of mRNA was not caused by mutations affecting transcriptional regulation. The ratio of the amount of neurofibromin to that of p120 GAP did not seem to be correlated with the extent of unequal allelic expression.

A new multisequence family in human.

By hybridizing total human DNA with probes derived from the extrachromosomal circular DNA fraction of cultured cells, we detected a human multisequence family, called chAB4, previously unknown. Approximately 50 copies of this sequence are located in the haploid human genome. The repetition units of chAB4 are 35 kb long and the units are tandemly arranged. DNA sequence analysis of parts of the chAB4 unit revealed no direct evidence for a possible function of the family, but possibly chAB4 harbors a gene. Family members are located on human chromosomes 1, 3, and 9 and on the short arms of chromosomes 13-15, 21, and 22. Therefore, in addition to the rDNA, chAB4 is the second class of clustered repetitive sequences with a relatively long repetition unit localized on the short arms of all acrocentric chromosomes. Some evolutionary aspects arising from the structure of chAB4, the established parts of its DNA sequences, and the chromosomal localization of this new multisequence family are discussed.

Long-range sequence composition mirrors linkage disequilibrium pattern in a 1.13 Mb region of human chromosome 22.

Association studies, the most powerful tool for the identification of genes underlying complex traits, depend on the observation of linkage disequilibrium (LD) between marker alleles and the trait. The LD pattern of the human genome which determines the regional density of required markers is non-uniform, with regions of long-range LD over several hundred kilobases and regions where LD extends only over a few kilobases. Studying LD in the NF1 gene region we encountered a transition from long-range to short-range LD which coincides with a switch in the isochore pattern. This observation prompted us to investigate the regional variation in the extent of LD more systematically and we selected an isochore transition within the MN1/PITPNB gene region on chromosome 22q12.1. Long-range LD characterizes the GC-poor (40% GC) parts of the sequences. No LD can be observed between closely spaced markers throughout the whole range of the GC-rich (50% GC) parts. In both cases, the NF1 and the MN1/PITPNB gene region, a clear-cut transition of the long-range GC content precisely coincides with a change in the extent of observable LD. The results can be explained by a 72-fold lower recombination frequency in the GC-poor, compared to the GC-rich isochores. Although recombination is not the only factor governing LD, our findings can help to predict levels of LD and marker densities required for association studies on the basis of regional GC content.