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Results for “ectopic gene conversion”

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PubMed1997

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

Cell Cycle↗

PubMed2002

Activation tagging of the two closely linked genes LEP and VAS independently affects vascular cell number.

The complex dominant Arabidopsis thaliana mutant lettuce (let) shows the conversion of the leaf petiole into a leaf blade caused by an ectopic leaf blade formation. This is the result of the activation tagging of the LEAFY PETIOLE (LEP) gene encoding an AP2/EREBP-like transcription factor. Here, we report that in addition to this leafy petiole phenotype, the size of the vascular bundles is increased in all aerial organs in let as a result of an increase in the number of xylem, phloem (pro)cambial and pericycle cells. This vascular phenotype is caused by activation tagging of the two genes VASCULAR TISSUE SIZE (VAS) and LEP. These genes are closely linked and arranged in tandem. Activation tagging of LEP only caused a specific increase in the number of xylem cells. This increased xylem cell number, together with the ectopic leaf blade formation, indicates that LEP functions as a cell division-promoting factor. The activation tagging of VAS only resulted in a specific increase in phloem (pro)cambial and pericycle cells. We conclude that activation tagging of LEP and VAS results in additive phenotypes. Insertional mutants for LEP and VAS display wild-type vascular development, indicating the relevance of activation tagging for functional analysis of novel genes involved in plant development.

Amino Acid Sequence↗

PubMed2003

Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas.

Germ-line mutations in the genes encoding succinate dehydrogenase complex subunits B (SDHB) and D (SDHD) have been reported in familial paragangliomas and apparently sporadic phaeochromocytomas (ASP), but the genotype-phenotype relationships of these mutations are unknown. Eighty-four patients (all but 2 followed up for 8.8 +/- 5.7 years) with ASP (57 with adrenal tumors, 27 with extra-adrenal, multiple, malignant, or recurrent tumors) were screened for the major susceptibility genes for phaeochromocytoma (RET, VHL, SDHD, and SDHB). Thirty-three tumors were available for molecular analysis, enzyme assays, and immunohistochemistry. No (0%) RET and 2 (2.4%) VHL mutations were detected. Only two coding single nucleotide polymorphisms in the SDHD gene (G12S and H50R) were found in 6 patients (7%). Conversely, six deleterious mutations in the SDHB gene were identified in 8 patients (9.5%). Ectopic site and recurrence or malignancy were strongly associated with SDHB mutations (7 of 8, 87%, versus 20 of 76, 26%; P = 0.001). Somatic DNA analysis indicated a loss of heterozygosity at chromosome 1p36 (SDHB locus) in 16 of 33 cases (48%). A loss of heterozygosity at the SDHB locus was found in all tumors with SDHB mutation, and assays of respiratory chain enzymes showed a complete loss of complex II catalytic activity. The vascular architecture of tumors with SDHB mutations displayed features typical of malignancy. These data strongly suggest that SDHB gene is a tumor suppressor gene and that the identification of germ-line mutations in SDHB gene in patients with ASPs should be considered as a high-risk factor for malignancy or recurrence.

Adrenal Gland Neoplasms↗

PubMed1996

Cyclin-mediated inhibition of muscle gene expression via a mechanism that is independent of pRB hyperphosphorylation.

It was recently demonstrated that ectopic expression of cyclin D1 inhibits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. Chem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cyclin D1 inhibits muscle gene expression without affecting MyoD DNA binding activity. Ectopic expression of cyclin D1 inhibits muscle gene activation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absent. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediated inhibition of myogenesis was due to hyperphosphorylation of pRB. In pRB-deficient fibroblasts, the ability of MyoD to activate the expression of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expression. The negative effects of cyclins A or E on muscle gene expression are, however, reversed by the presence of a mutated form of pRB which cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit muscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G1 cyclin-cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphosphorylation and one that is independent of pRB hyperphosphorylation.

Animals↗

PubMed2002

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L).

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

Cells, Cultured↗

PubMed2003

Complex evolution of 7E olfactory receptor genes in segmental duplications.

Large segmental duplications (SDs) constitute at least 3.6% of the human genome and have increased its size, complexity, and diversity. SDs can mediate ectopic sequence exchange resulting in gross chromosomal rearrangements that could contribute to speciation and disease. We have identified and evaluated a subset of human SDs that harbor an 88-member subfamily of olfactory receptor (OR)-like genes called the 7Es. At least 92% of these genes appear to be pseudogenes when compared to other OR genes. The 7E-containing SDs (7E SDs) have duplicated to at least 35 regions of the genome via intra- and interchromosomal duplication events. In contrast to many human SDs, the 7E SDs are not biased towards pericentromeric or subtelomeric regions. We find evidence for gene conversion among 7E genes and larger sequence exchange between 7E SDs, supporting the hypothesis that long, highly similar stretches of DNA facilitate ectopic interactions. The complex structure and history of the 7E SDs necessitates extension of the current model of large-scale DNA duplication. Despite their appearance as pseudogenes, some 7E genes exhibit a signature of purifying selection, and at least one 7E gene is expressed.

Amino Acid Sequence↗

PubMed2003

Bowl is required downstream of Notch for elaboration of distal limb patterning.

In the Drosophila leg, activation of Notch leads to the establishment of the joints that subdivide the appendage into segments. We find that mutations in bowl result in similar phenotypes to Notch, causing fusion and truncations of tarsal segments (tarsomeres) and, like its close relative Odd-skipped, Bowl is produced in response to Notch signalling at a subset of segment boundaries. However, despite the fact that bowl mutant clones result in fusion of tarsomeres, Bowl protein is only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere joints. One hypothesis to reconcile these data is that bowl has a role at an earlier stage in tarsal development. We therefore investigated the effects of bowl mutations on the expression of leg 'gap' genes that confer regional identity on the developing leg. Several of these genes have altered expression in bowl mutant cells. For example, bric-a-brac2 is normally expressed in the central part of the tarsus domain but expands into distal and proximal regions in bowl clones. Conversely, ectopic bowl leads to a reduction in bric-a-brac2, with a concomitant expansion of proximal (t1) and distal (t5) tarsomere fates. The bowl gene is therefore required for the elaboration of pattern in the tarsus and its effects suggest a progressive model for the determination of P/D identities. This mechanism might be important in the diversification of arthropod limbs, because it explains how segmented tarsomeres could have arisen from an ancestral limb with an unsegmented tarsus.

Animals↗

PubMed1997

Oncogenic transformation and inhibition of adipocytic conversion of preadipocytes by TLS/FUS-CHOP type II chimeric protein.

Myxoid liposarcomas are characterized by t(12; 16)(q13;p11) translocation and expression of TLS/ FUS-CHOP chimeric transcripts (types I to III). Among these, the type II transcript is expressed in the majority of cases of myxoid and round cell liposarcoma. To investigate the function of the type II chimeric protein, we obtained stable transformants of ST-13, a murine preadipocytic cell line, which express TLS/FUS-CHOP type II protein (ST-TC) or CHOP protein (ST-C) as well as vector-transfected controls (ST-V). ST-TC and ST-C cells showed almost complete or partial resistance to adipogenic conversion by insulin and thiazolidinedione, respectively. Induction by adipogenic stimulation of the adipocytic genes such as C/EBP alpha, aP2, and adipsin was almost totally suppressed in the ST-TC cells, whereas in ST-C cells C/EBP alpha alone was induced without induction of aP2 and adipsin. Transcriptional suppression of the C/EBP alpha gene in ST-TC cells was suggested by the results of chloramphenicol acetyltransferase (CAT) assay showing a significantly lower C/EBP alpha promoter activity compared with findings in ST-C and ST-V cells. Failure to rescue adipogenic conversion by ectopic expression of C/EBP alpha in ST-TC cells suggested a functional impairment of C/EBP alpha to induce expression of downstream genes. TLS/FUS-CHOP type II protein showed transforming activity, as evidenced by loss of contact inhibition of growth, anchorage-independent growth in soft agar, and tumor formation in nude mice, showing typical histological features of myxoid liposarcoma seen in humans. These findings suggest important roles for TLS/FUS-CHOP type II protein in the oncogenesis of myxoid liposarcoma.

Adipocytes↗

PubMed2005

Convergent genetic programs regulate similarities and differences between related motor neuron classes in Caenorhabditis elegans.

How do genetic programs create features common to a specific cell or tissue type while generating modifications necessary for functional diversification? We have addressed this question using the nematode Caenorhabditis elegans. The dorsal D (DD) and ventral D (VD) motorneurons (mns), referred to collectively as the D mns, compose a cross-inhibitory network that contributes to the animal's sinuous locomotion. The D mns share a number of structural and functional features, but are distinguished from one another by their synaptic patterns and the expression of a neuropeptide gene. Our findings suggest that the similarities and differences are generated at the transcriptional level. UNC-30 contains a homeodomain and activates structural and functional genes expressed in both classes. UNC-55 is a nuclear receptor expressed in the VD mns that is necessary for generating features that distinguish the two classes of D mns from one another. In unc-55 mutants, the VD mns adopt the DD mn synaptic pattern and peptide expression profile. Conversely, ectopic expression of unc-55 in the DD mns causes them to adopt VD mn features. The promoter of the neuropeptide gene expressed in the DD mns contains putative binding sites for both UNC-30 and UNC-55; alteration of these sites suggests that UNC-55 represses the ability of UNC-30 to activate a subset of genes that are expressed in the DD mns but not in the VD mns. Thus UNC-55 acts as a switch for the features that distinguish these two functionally related classes of mns.

Amino Acid Motifs↗

PubMed2002

Nk6, a novel Drosophila homeobox gene regulated by vnd.

Nk(x)-type homeobox genes are an evolutionarily conserved family that regulate diverse developmental processes. Here we describe a novel Drosophila gene, Nk6, which encodes an Nk-type transcription factor most homologous to vertebrate Nkx6.1 and Nkx6.2. The homeodomains and NK decapeptide domains of all three proteins are highly conserved. Nk6 is expressed in the embryonic brain, ventral nerve cord, hindgut, and internal head structures. Nerve cord expression is in midline precursors, several ventral and intermediate column neuroblasts, and later in neurons but not glia, similar to the known expression of Nkx6 genes in the neural tube. We show genetically that Nk6 is positively regulated, directly or indirectly, by vnd in brain precursors. In vnd mutants, head neuroectoderm Nk6 expression is abolished where it is normally co-expressed with vnd. Conversely, vnd-overexpression leads to ectopic Nk6 expression in the brain. These findings further highlight the importance of interactions between Nk(x)-type genes in regulating their expression.

Amino Acid Sequence↗

PubMed2005

Neuromancer Tbx20-related genes (H15/midline) promote cell fate specification and morphogenesis of the Drosophila heart.

The Tbx family of transcription factors are prominently expressed in the early cardiac primordium throughout the animal kingdom. Mutations in Tbx genes result invariably in defective formation and function of the heart, including congenital heart disease in humans. Similar to their vertebrate counterpart, the Drosophila Tbx20 gene pair, neuromancer1 (nmr1, FlyBase:H15) and neuromancer2 (nmr2, Flybase:mid), exhibits a dynamic expression pattern, including in all contractile myocardial cells. Deletion mutants of nmr1 combined with mesoderm-specific knock-down of nmr2 exhibit phenotypes that suggest nmr is critical for correct specification of the cardiac progenitor populations as well as for morphogenesis and assembly of the contractile heart tube. Loss-of-nmr-function causes a switch in cell fates in the cardiogenic region, in that the progenitors expressing the homeobox gene even skipped (eve) are expanded accompanied by a corresponding reduction of the progenitors expressing the homeobox gene ladybird (lbe). As a result, the number of differentiating myocardial cells is severely reduced whereas pericardial cell populations are expanded. Conversely, pan-mesodermal expression of nmr represses eve, while causing an expansion of cardiac lbe expression, as well as ectopic mesodermal expression of the homeobox gene tinman. In addition, nmr mutants with less severe penetrance exhibit cell alignment defects of the myocardium at the dorsal midline, suggesting nmr is also required for cell polarity acquisition of the heart tube. In exploring the regulation of nmr, we find that the GATA factor Pannier is essential for cardiac expression, and acts synergistically with Tinman in promoting nmr expression. Moreover, reducing nmr function in the absence of pannier further aggravates the deficit in cardiac mesoderm specification. Taken together, the data suggest that nmr acts both in concert with and subsequent to pannier and tinman in cardiac specification and differentiation. We propose that nmr is another determinant of cardiogenesis, along with tinman and pannier.

Animals↗

PubMed2006

Allelic recombination and de novo deletions in sperm in the human beta-globin gene region.

Meiotic recombination is of fundamental importance in creating haplotype diversity in the human genome and has the potential to cause genomic rearrangements by ectopic recombination between repeat sequences and through other changes triggered by recombination-initiating events. However, the relationship between allelic recombination and genome instability in the human germline remains unclear. We have therefore analysed recombination and DNA instability in the delta-, beta-globin gene region and its associated recombination hotspot. Sperm typing has for the first time accurately defined the hotspot and shown it to be the most active autosomal crossover hotspot yet described, although unusually inactive in non-exchange gene conversion. The hotspot just extends into a homology block shared by the delta- and beta-globin genes, within which ectopic exchanges can generate Hb Lepore deletions. We developed a physical selection method for recovering and validating extremely rare de novo deletions in human DNA and used it to characterize the dynamics of these Hb Lepore deletions in sperm as well as other deletions not arising from ectopic exchanges between homologous DNA sequences. Surprisingly, both classes of deletion showed breakpoints that avoided the beta-globin hotspot, establishing that it possesses remarkable fidelity and does not play a significant role in triggering these DNA rearrangements. This study also provides the first direct analysis of de novo deletion in the human germline and points to a possible deletion-controlling element in the beta-globin gene separate from the crossover hotspot.

Alleles↗

PubMed2002

AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts.

Activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is indispensable for somatic hypermutation (SHM), class switch recombination, and gene conversion of immunoglobulin genes, which indicates a common molecular mechanism for these phenomena. Here we show that ectopic expression of AID alone can induce hypermutation in an artificial GFP substrate in NIH 3T3 murine fibroblast cells. The frequency of mutations was closely correlated with the level of transcription of the target gene, and the distribution of mutations in NIH 3T3 cells was similar to those of SHM in B lymphocytes. These results indicate that AID is sufficient for the generation of SHM in an actively transcribed gene in fibroblasts, as well as B cells, and that any of the required cofactors must be present in these fibroblasts.

3T3 Cells↗

PubMed2002

The mouse polydactylous mutation, luxate (lx), causes anterior shift of the anteroposterior border in the developing hindlimb bud.

Pattern formation along the anterior-posterior axis of the vertebrate limb is established upon activation of Sonic Hedgehog (SHH) in the zone of polarizing activity (ZPA). Since many mouse mutants with preaxial polydactyly show ectopic expression of Shh at the anterior margin of the limb buds, it has been thought to be a primary defect caused by these mutations. We show here that the mouse mutation luxate (lx) exhibits dose-dependent reduction in the size of the Fgf8 expression domain in the ectoderm from the initial stage of limb development. This aberration was independent of Fgf10 expression in the limb mesenchyme. Shh was induced in the mesenchyme underlying the posterior end of the Fgf8 expression domain, indicating an anterior shift of Shh expression in lx hindlimb buds. Prior to the ectopic induction of Shh, the expression domains of genes downstream from Shh, namely dHAND, Gli1, Ptc and Gre, which are normally expressed in posterior mesenchyme of limb buds, expanded anteriorly on the lx hindlimb buds. Conversely, the expression domains of anterior mesenchymal markers such as Gli3and Alx4 decreased in size. Thus, ectopic Shh is not a primary defect of the lx mutation. Rather, our results indicate that the lx mutation affects the positioning of the anteroposterior border in developing hindlimb buds.

Animals↗

PubMed1992

Skeletal muscle phenotypes initiated by ectopic MyoD in transgenic mouse heart.

Forced expression of the myogenic regulatory gene MyoD in many types of cultured cells initiates their conversion into skeletal muscle. It is not known, however, if MyoD expression serves to activate all or part of the skeletal muscle program in vivo during animal development, nor is it known how limiting the influences of cellular environment may be on the regulatory effects of MyoD. To begin to address these issues, we have produced transgenic mice which express MyoD in developing heart, where neither MyoD nor its three close relatives--myogenin, Myf-5, and MRF4/herculin/Myf-6--are normally expressed. The resulting gross phenotype in offspring from multiple, independent transgenic founders includes abnormal heart morphology and ultimately leads to death. At the molecular level, affected hearts exhibit activation of skeletal muscle-specific regulatory as well as structural genes. We conclude that MyoD is able to initiate the program that leads to skeletal muscle differentiation during mouse development, even in the presence of the ongoing cardiac differentiation program. Thus, targeted misexpression of this tissue-specific regulator during mammalian embryogenesis can activate, either directly or indirectly, a diverse set of genes normally restricted to a different cell lineage and a different cellular environment.

Animals↗

PubMed2004

Drosophila cardiac tube organogenesis requires multiple phases of Hox activity.

The segmented Drosophila linear cardiac tube originates from two cell lineages that give rise to the anterior aorta (AA) and the posterior cardiac tube. The three Hox genes of the Bithorax Complex as well as Antennapedia (Antp) have been shown to be expressed in the posterior cardiac tube, while no Hox gene is expressed in the anterior aorta. We show that the cells of the whole tube adopt the anterior aorta identity in the complete absence of Hox function. Conversely, ectopic expression of Antp, Ultrabithorax (Ubx), or abdominal-A (abd-A) transformed the anterior aorta into posterior cardiac tube by all available criteria, indicating an equivalent early function in their ability to direct a posterior cardiac tube lineage. We further demonstrate that Hox genes act in a subsequent step during cardiac tube organogenesis, specifically on the differentiation of posterior cardiac tube myocytes. In addition, while some of these functions are fulfilled equally well by any one of the three Hox genes, some others are specific to a given Hox. Notably, the gene encoding the anion transporter Na+-Driven Anion Exchanger 1 behaves as a Hox differential transcriptional target and is activated by abd-A in the heart and repressed by Ubx in the posterior aorta. This analysis illustrates the mechanisms by which Hox genes can orchestrate organogenesis and, in particular, allows a clear uncoupling of the different phases of Hox activity in this process.

Animals↗

PubMed2005

Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm.

The molecular basis of vertebrate germ layer formation has been the focus of intense scrutiny for decades, and the inductive interactions underlying this process are well defined. Only recently, however, have studies demonstrated that the regulated inhibition of ectopic germ layer formation is also crucial for patterning the early vertebrate embryo. We report here the characterization of Xema (Xenopus Ectodermally-expressed Mesendoderm Antagonist), a novel member of the Foxi-subclass of winged-helix transcription factors that is involved in the suppression of ectopic germ layer formation in the frog, Xenopus laevis. Xema transcripts are restricted to the animal pole ectoderm during early Xenopus development. Ectopic expression of Xema RNA inhibits mesoderm induction, both by growth factors and in the marginal zone, in vivo. Conversely, introduction of antisense morpholino oligonucleotides directed against the Xema transcript stimulates the expression of a broad range of mesodermal and endodermal marker genes in the animal pole. Our studies demonstrate that Xema is both necessary and sufficient for the inhibition of ectopic mesendoderm in the cells of the presumptive ectoderm, and support a model in which Fox proteins function in part to restrict inappropriate germ layer development throughout the vertebrate embryo.

Amino Acid Sequence↗

PubMed2003

Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development.

The respiratory primordium is positioned and its territory is defined in the foregut. The visceral mesoderm of the respiratory primordium acquires the inducing potential that is necessary for endodermal budding morphogenesis and respiratory endoderm formation. Tbx4, a member of the T-box transcription factor gene family, was specifically expressed in the visceral mesoderm of the lung primordium. To analyze the function of Tbx4, we ectopically expressed Tbx4 in the visceral mesoderm of the foregut using in ovo electroporation. Ectopic Tbx4 induced ectopic bud formation in the esophagus by activating the expression of Fgf10. Conversely, interference of Tbx4 function resulted in repression of Fgf10 expression and in failure of lung bud formation. In addition, ectopic Tbx4 or Fgf10 also induced ectopic expression of Nkx2.1, a marker gene specific for the respiratory endoderm, in the underlying esophagus endoderm. When the border of the Tbx4 expression domain, which demarcates the respiratory tract and the esophagus, was disturbed by misexpression of Tbx4, formation of the tracheo-esophageal septum failed. These results suggested that Tbx4 governs multiple processes during respiratory tract development; i.e. the initial endodermal bud formation, respiratory endoderm formation, and septation of the respiratory tract and the esophagus.

Animals↗