Evidence for at least four Fanconi anaemia genes including FACC on chromosome 9.

Fanconi anaemia (FA) is a DNA repair disorder characterized by cellular hypersensitivity to DNA cross-linking agents and extensive phenotypic heterogeneity. To determine the extent of genetic heterogeneity present in FA, a panel of somatic cell hybrids was constructed using polyethylene glycol-mediated cell fusion. Three new complementation groups were identified, designated FA(B), FA(C) and FA(D), and the gene defective in FA(C) which we have recently cloned was localized to chromosome 9q22.3 through in situ hybridization. These results suggest that mutations in at least four different genes lead to FA, a degree of genetic heterogeneity comparable to that of other DNA repair disorders.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Complex tools and motor-to-mechanical transformations.

The ability to use complex tools is thought to depend on multifaceted motor-to-mechanical transformations within the left inferior parietal lobule (IPL), linked to cognitive control over compound actions. Here we show using neuroimaging that demanding transformations of finger movements into proper mechanical movements of functional parts of complex tools invoke significantly the right rather than left rostral IPL, and bilateral posterior-to-mid and left anterior intraparietal sulci. These findings emerged during the functional grasp and tool-use programming phase. The expected engagement of left IPL was partly revealed by traditional region-of-interest analyses, and further modeling/estimations at the hand-independent level. Thus, our results point to a special role of right IPL in supporting sensory-motor spatial mechanisms which enable an effective control of fingers in skillful handling of complex tools. The resulting motor-to-mechanical transformations involve dynamic hand-centered to target-centered reference frame conversions indispensable for efficient interactions with the environment.

Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1.

The Fanconi anemia group C protein (FANCC) plays an important role in hematopoiesis by ensuring the survival of hematopoietic progenitor cells through an unknown mechanism. We investigated the function of FANCC by identifying FANCC-binding proteins in hematopoietic cells. Here we show that glutathione S-transferase P1-1 (GSTP1) interacts with FANCC, and that overexpression of both proteins in a myeloid progenitor cell line prevents apoptosis following factor deprivation. FANCC increases GSTP1 activity after the induction of apoptosis. GSTP1 is an enzyme that catalyzes the detoxification of xenobiotics and by-products of oxidative stress, and it is frequently upregulated in neoplastic cells. Although FANCC lacks homology with conventional disulfide reductases, it functions by preventing the formation of inactivating disulfide bonds within GSTP1 during apoptosis. The prevention of protein oxidation by FANCC reveals a novel mechanism of enzyme regulation during apoptosis and has implications for the treatment of degenerative diseases with thiol reducing agents.

Killing of fibroblasts by dexamethasone or dibutyryl adenosine 3',5'-monophosphate is not a valid test for cystic fibrosis.

Assays based on the counting of total cells and of colony-forming cells were used to demonstrate that neither dexamethasone nor dibutyryl adenosine 3',5'-monophosphate (cyclic AMP) kills human fibroblasts under a variety of conditions. These results contradict those of previous studies showing that dexamethasone and dibutyryl cyclic AMP kill a higher percentage of fibroblasts from normal humans than from individuals with cystic fibrosis.

Identification of the cystic fibrosis gene: genetic analysis.

Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.

Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker.

A polymorphic DNA marker has been found genetically linked, in a set of 39 human families, to an autosomal recessive gene that causes cystic fibrosis (CF), a disease affecting one in 2000 Caucasian children. The DNA marker (called D0CRI-917) is also linked to the PON locus, which by independent evidence is linked to the CF locus. The best estimates of the genetic distances are 5 centimorgans between the DNA marker and PON and 15 centimorgans between the DNA marker and the CF locus, meaning that the location of the disease gene has been narrowed to about 1 percent of the human genome (about 30 million base pairs). Although the data are consistent with the interpretation that a single locus causes cystic fibrosis, the possibility of genetic heterogeneity remains. The discovery of a linked DNA polymorphism is the first step in molecular analysis of the CF gene and its causative role in the disease.

Mammalian DNA-repair genes.

The sequence and functional homology of certain genes between mammalian and non-mammalian eukaryotes has facilitated significant advances in our understanding of mammalian DNA repair. Several novel DNA damage and repair genes have been identified by using a variety of approaches. Study of these genes will lead to an increased understanding of the biological consequences of aberrant DNA maintenance in humans and other species.

Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells.

BACKGROUND: Many differentiating tissues contain progenitor cells that differ in their commitment states but cannot be readily distinguished or segregated. Molecular analysis is therefore restricted to mixed populations or cell lines which may also be heterogeneous, and the critical differences in gene expression that might determine divergent development are obscured. In this study, we combined global amplification of mRNA transcripts in single cells with identification of the developmental potential of processed cells on the basis of the fates of their sibling cells from clonal starts. RESULTS: We analyzed clones of from four to eight hemopoietic precursor cells which had a variety of differentiative potentials; sibling cells generally each formed clones of identical composition in secondary culture. Globally amplified cDNA was prepared from individual precursors whose developmental potential was identified by tracking sibling fates. Further cDNA samples were prepared from terminally maturing, homogeneous hemopoietic cell populations. Together, the samples represented 16 positions in the hemopoietic developmental hierarchy. Expression patterns in the sample set were determined for 29 genes known to be involved in hemopoietic cell growth, differentiation or function. The cDNAs from a bipotent erythroid/megakaryocyte precursor and a bipotent neutrophil/macrophage precursor were subtractively hybridized, yielding numerous differentially expressed cDNA clones. Hybridization of such clones to the entire precursor sample set identified transcripts with consistent patterns of differential expression in the precursor hierarchy. CONCLUSIONS: Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

Induction of Fanconi anemia cellular phenotype in human 293 cells by overexpression of a mutant FAC allele.

The polypeptide encoded by the Fanconi anemia (FA) complementation group C gene, FAC, binds to a group of cytoplasmic proteins in vitro and may form a multimeric complex. A known mutant allele of FAC resulting from the substitution of Pro for Leu at codon 554 fails to correct the sensitivity of FA group C cells to mitomycin C. We reasoned that overexpression of the mutant protein in a wild-type cellular background might induce the FA phenotype by competing with endogenous FAC for binding to the accessory proteins. After stable transfection of 293 cells with wild-type and a mutant FAC allele containing the L554P substitution, four independent clones that expressed four-to-fifteen fold higher levels of transcript from the mutant transgene relative to the endogenous FAC gene showed hypersensitivity to mitomycin C. By contrast, both parental and FAC-overexpressing cells maintained their relative resistance to mitomycin C. No differences in the biosynthesis, subcellular localization and protein interactions of the normal and mutant proteins were detected. The induction of the FA phenotype in this system is compatible with the competition hypothesis and provides support for a functional role of the FAC-binding proteins in vivo.

Lung disease in mice with cystic fibrosis.

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents.

In order to develop the usefulness of Fanconi's anemia (FA) lymphoblast lines for biochemical and genetic studies, we have determined their sensitivity to a variety of DNA-damaging chemicals. We have adapted a growth inhibiton protocol in which the sensitivity of a cell line is characterized by the drug concentration yielding a 50% inhibiton of growth (EC50). The DNA-cross-linking agents, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis-diaminedichloroplatinum(II), and cyclophosphamide, were all more toxic to four FA cell lines than to five normal lines. Three lines, HSC 72 (FA), 99 (FA) and 230 (FA), had EC50s that were 10 to 20 times lower than that of controls while the fourth line, HSC 62 (FA), had an intermediate EC50. Three nitrosourea compounds were also more toxic to FA cells than to controls. However, 2 normal cell lines (HSC 92 and 93) had nitrosourea EC50s 4 to 7 times lower than the other nine controls and overlapped the sensitivity of the intermediate [HSC 62 (FA)] cell line. The same 2 normal cell lines were also more sensitive than 12 other controls, including FA heterozygotes, xeroderma pigmentosum, and ataxia telangiectasis, to the monofunctional alkylating agents, ethyl methane sulfonate, methyl methane sulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine. Heterogeneity was also found with FA lines. Two FA cell lines (HSC 72 and 230) had EC50s lower than all control lines while one FA line (HSC 99) had an EC50 similar to that of the resistant normal lines. FA and normal cells had nearly the same sensitivity to 4-nitroquinoline-1-oxide and bleomycin. These results demonstrate that FA lymphoblast lines are more sensitive than normal cell lines to all DNA-cross-linking agents examined. These cell lines should therefore be useful for the analysis of DNA cross-link repair and the biochemical defect in FA. We have also found an unexpected sensitivity of some FA and normal lines to monofunctional alkylating agents.