HC Forum®: a web site based on an international human cytogenetic database

Familial structural rearrangements of chromosomes represent a factor of malformation risk that could vary over a large range, making genetic counseling difficult. However, they also represent a powerful tool for increasing knowledge of the genome, particularly by studying breakpoints and viable imbalances of the genome. We have developed a collaborative database that now includes data on more than 4100 families, from which we have developed a web site called HC Forum® (http://HCForum.imag.fr). It offers geneticists assistance in diagnosis and in genetic counseling by assessing the malformation risk with statistical models. For researchers, interactive interfaces exhibit the distribution of chromosomal breakpoints and of the genome regions observed at birth in trisomy or in monosomy. Dedicated tools including an interactive pedigree allow electronic submission of data, which will be anonymously shown in a forum for discussions. After validation, data are definitively registered in the database with the email of the sender, allowing direct location of biological material. Thus HC Forum® constitutes a link between diagnosis laboratories and genome research centers, and after 1 year, more than 700 users from about 40 different countries already exist.

Mechanisms underlying losses of heterozygosity in human colorectal cancers

Losses of heterozygosity are the most common molecular genetic alteration observed in human cancers. However, there have been few systematic studies to understand the mechanism(s) responsible for losses of heterozygosity in such tumors. Here we report a detailed investigation of the five chromosomes lost most frequently in human colorectal cancers. A total of 10,216 determinations were made with 88 microsatellite markers, revealing 245 chromosomal loss events. The mechanisms of loss were remarkably chromosome-specific. Some chromosomes displayed complete loss such as that predicted to result from mitotic nondisjunction. However, more than half of the losses were associated with losses of only part of a chromosome rather than a whole chromosome. Surprisingly, these losses were due largely to structural alterations rather than to mitotic recombination, break-induced replication, or gene conversion, suggesting novel mechanisms for the generation of much of the aneuploidy in this common tumor type.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the Ldlr knockout mouse model

Atherosclerosis is a complex disease resulting from the interaction of multiple genes. We have used the Ldlr knockout mouse model in an interspecific genetic cross to map atherosclerosis susceptibility loci. A total of 174 (MOLF/Ei × B6.129S7-Ldlrtm1Her) × C57BL/6J-Ldlrtm1Her backcross mice, homozygous for the Ldlr null allele, were fed a Western-type diet for 3 months and then killed for quantification of aortic lesions. A genome scan was carried out by using DNA pools and microsatellite markers spaced at ≈18-centimorgan intervals. Quantitative trait locus analysis of individual backcross mice confirmed linkages to chromosomes 4 (Athsq1, logarithm of odds = 6.2) and 6 (Athsq2, logarithm of odds = 6.7). Athsq1 affected lesions in females only whereas Athsq2 affected both sexes. Among females, the loci accounted for ≈50% of the total variance of lesion area. The susceptible allele at Athsq1 was derived from the MOLF/Ei genome whereas the susceptible allele at Athsq2 was derived from C57BL/6J. Inheritance of susceptible alleles at both loci conferred a 2-fold difference in lesion area, suggesting an additive effect of Athsq1 and Athsq2. No associations were observed between the quantitative trait loci and levels of plasma total cholesterol, high density lipoprotein cholesterol, non-high density lipoprotein cholesterol, insulin, or body weight. We provide strong evidence for complex inheritance of atherosclerosis in mice with elevated plasma low density lipoprotein cholesterol and show a major influence of nonlipoprotein-related factors on disease susceptibility. Athsq1 and Athsq2 represent candidate susceptibility loci for human atherosclerosis, most likely residing on chromosomes 1p36–32 and 12p13–12, respectively.

Characterization of a Novel Human SMC Heterodimer Homologous to the Schizosaccharomyces pombe Rad18/Spr18 Complex

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

A bacterial artificial chromosome-based framework contig map of human chromosome 22q.

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.

Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes.

Chédiak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melanosome morphology and function. We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation. The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418. When YAC-complemented beige cells were fused to human Chédiak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred. These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

Human TOP3: a single-copy gene encoding DNA topoisomerase III.

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12.

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis.