Homeotic Arm-to-Leg Transformation Associated with Genomic Rearrangements at the PITX1 Locus

The study of homeotic-transformation mutants in model organisms such as Drosophila revolutionized the field of developmental biology, but how these mutants relate to human developmental defects remains to be elucidated. Here, we show that Liebenberg syndrome, an autosomal-dominant upper-limb malformation, shows features of a homeotic limb transformation in which the arms have acquired morphological characteristics of a leg. Using high-resolution array comparative genomic hybridization and paired-end whole-genome sequencing, we identified two deletions and a translocation 5′ of PITX1. The structural changes are likely to remove active PITX1 forelimb suppressor and/or insulator elements and thereby move active enhancer elements in the vicinity of the PITX1 regulatory landscape. We generated transgenic mice in which PITX1 was misexpressed under the control of a nearby enhancer and were able to recapitulate the Liebenberg phenotype.     

Genomic Rearrangements in Neisseria meningitidis Strains of the ET-5 Complex

A physical map of the chromosome of Neisseria meningitidis strain 44/76, which belongs to the epidemic clone ET-5, was constructed. DNA fragments obtained after SfiI and NheI digestion were resolved by pulsed field gel electrophoresis (PFGE). The overall arrangement of 26 genetic markers localized on the 2.3-Mb chromosome was conserved in comparison with that in meningococcal strains B1940 and Z2491. Simplified physical maps of 29 additional strains belonging to the ET-5 complex isolated from various parts of the world were compared with that of strain 44/76. Ten distinct patterns of hybridization were identified. While two of the seven probes hybridized to fragments of the same size in all strains, the remaining probes hybridized to different fragments, in some cases to fragments not adjacent on the chromosome of 44/76. These results indicated the occurrence of genetic rearrangements in the genome of the ET-5 meningococcal clone in the course of its epidemic spread. Received: 17 November 1999 / Accepted: 28 December 1999     

P Element Insertions and Rearrangements at the Singed Locus of Drosophila Melanogaster

DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.     

Preemptive Homology-Directed DNA Repair Fosters Complex Genomic Rearrangements in Hepatocellular Carcinoma

Degree of genomic instability closely correlates with poor prognosis, drug resistance as well as poor survival across human cancer of different origins. This study assessed the relationship between DNA damage response (DDR) and chromosome instability in hepatocellular carcinoma (HCC). We investigated DDR signaling in HCC cells by analyzing DNA damage-dependent redistribution of major DDR proteins to damaged chromatin using immunofluorescence microscopy and Western blotting experimentations. We also performed gene conversion and metaphase analyses to address whether dysregulated DDR may bear any biological significance during hepatocarcinogenesis. Accordingly, we found that HCC cell lines suffered from elevated spontaneous DNA double-strand breaks (DSBs). In addition, analyses of HCC metaphases revealed marked aneuploidy and frequent sister chromatid exchanges when compared to immortalized hepatocytes, the latter of which were further induced following camptothecin-induced DSBs. We propose that genomic instability in HCC may be caused by erroneous DNA repair in a desperate attempt to mend DSBs for cell survival and that such preemptive measures inadvertently foster chromosome instability and thus complex genomic rearrangements.     

Cloning the Arabidopsis GA1 Locus by Genomic Subtraction

Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.     

Epigenetic Regulation of Programmed Genomic Rearrangements in Paramecium aurelia1

ABSTRACT In ciliates, development of the polyploid somatic macronucleus after sexual events involves extensive and reproducible rearrangements of the germ-line genome, including chromosome fragmentation and precise excision of numerous internal sequence elements. In Paramecium aurelia, alternative macronuclear versions of the same germ-line genome can be maternally inherited across sexual generations, showing that rearrangement patterns are not strictly determined by the germ-line sequence. Homology-dependent maternal effects can be evidenced by transformation of the vegetative macronucleus with cloned macronuclear sequences: new fragmentation patterns or internal deletions are specifically induced during differentiation of a new macronucleus, in sexual progeny of transformed clones. Furthermore, transformation of the maternal macronucleus with germ-line sequences containing internal eliminated sequences (short single-copy elements) can result in a specific inhibition of the excision of the same elements in the zygotic macronucleus. These experiments show that the processing of many germ-line sequences in the developing macronucleus is sensitive to the structure and copy number of homologous sequences in the maternal macronucleus. The generality and sequence specificity of this trans-nuclear, epigenetic regulation of rearrangements suggest that it is mediated by pairing interactions between germ-line sequences and sequences imported from the maternal macronucleus.     

Mutation-Selection Balance under Genomic Imprinting at an Autosomal Locus

I model the effect of genomic imprinting on the equilibrium allele frequencies at an autosomal diallelic locus subject to viability selection and mutation. The population size is assumed to be very large; male and female mutation rates may be unequal. Different models examine cases of the inactivation of one gene (with both complete and partial penetrance) and of differential expression of genes according to the parent of origin. In the simplest cases the frequency of the deleterious allele is approximately twice that of a dominant nonimprinting mutant, but considerably less than that of a recessive nonimprinting mutant. Under imprinting, selection and unequal mutation rates interact: other things being equal, male-biased mutation leads to lower mutant frequencies under maternal imprinting and higher frequencies under paternal imprinting. I also model cases where just one allele is imprintable (and the other not). These models allow us to predict the frequency of a failure to imprint in a normally imprinting system, as well as the frequency of imprinting at a standard nonimprinting locus.     

Recombination and Other Genomic Rearrangements in Picornaviruses

The available data on rearrangements (recombinations, deletions, and insertions) of picornavirus genomes fit the replicative template switch model postulating that an incomplete nascent minus RNA strand leaves the template and resumes its synthesis on another template (or another locus of the original template). The nascent strand dissociation is believed to be facilitated by the elongation pausing caused by secondary structure elements or nucleotide misincorporations. Rearrangements may involve (nearly) identical or completely dissimilar pairs of parting and anchoring sites. Rearrangements contribute to both conservation and variation of the picornaviral genomes.     

A Non-Coding Genomic Duplication at the HMX1 Locus Is Associated with Crop Ears in Highland Cattle

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.     

Unraveling Genomic Complexity at a Quantitative Disease Resistance Locus in Maize

Multiple disease resistance has important implications for plant fitness, given the selection pressure that many pathogens exert directly on natural plant populations and indirectly via crop improvement programs. Evidence of a locus conditioning resistance to multiple pathogens was found in bin 1.06 of the maize genome with the allele from inbred line “Tx303” conditioning quantitative resistance to northern leaf blight (NLB) and qualitative resistance to Stewart’s wilt. To dissect the genetic basis of resistance in this region and to refine candidate gene hypotheses, we mapped resistance to the two diseases. Both resistance phenotypes were localized to overlapping regions, with the Stewart’s wilt interval refined to a 95.9-kb segment containing three genes and the NLB interval to a 3.60-Mb segment containing 117 genes. Regions of the introgression showed little to no recombination, suggesting structural differences between the inbred lines Tx303 and “B73,” the parents of the fine-mapping population. We examined copy number variation across the region using next-generation sequencing data, and found large variation in read depth in Tx303 across the region relative to the reference genome of B73. In the fine-mapping region, association mapping for NLB implicated candidate genes, including a putative zinc finger and pan1. We tested mutant alleles and found that pan1 is a susceptibility gene for NLB and Stewart’s wilt. Our data strongly suggest that structural variation plays an important role in resistance conditioned by this region, and pan1, a gene conditioning susceptibility for NLB, may underlie the QTL.     

Complex Signatures of Natural Selection at the Duffy Blood Group Locus

The Duffy blood group locus (FY) has long been considered a likely target of natural selection, because of the extreme pattern of geographic differentiation of its three major alleles (FY*B, FY*A, and FY*O). In the present study, we resequenced the FY region in samples of Hausa from Cameroon (fixed for FY*O), Han Chinese (fixed for FY*A), Italians, and Pakistanis. Our goals were to characterize the signature of directional selection on FY*O in sub-Saharan Africa and to understand the extent to which natural selection has also played a role in the extreme geographic differentiation of the other derived allele at this locus, FY*A. The data from the FY region are compared with the patterns of variation observed at 10 unlinked, putatively neutral loci from the same populations, as well as with theoretical expectations from the neutral-equilibrium model. The FY region in the Hausa shows evidence of directional selection in two independent properties of the data (i.e., level of sequence variation and frequency spectrum), observations that are consistent with the FY*O mutation being the target. The Italian and Chinese FY data show patterns of variation that are very unusual, particularly with regard to frequency spectrum and linkage disequilibrium, but do not fit the predictions of any simple model of selection. These patterns may represent a more complex and previously unrecognized signature of positive selection.     

Escape from Genomic Imprinting at the Mouse T-Associated Maternal Effect (Tme) Locus

Genomic imprinting occurs at the paternally inherited allele of the mouse T-associated maternal effect (Tme) locus. As a consequence, maternal transmission of a functional Tme gene is normally required for viability and individuals that receive a Tme-deleted chromosome (Thp or tlub2) from their mother die late in gestation or shortly thereafter. Here we report that a rearranged paternally derived chromosome duplicated for the Tme locus can act to rescue animals that have not received a maternal copy of the Tme locus. Unexpectedly, all rescued animals display an abnormal short/kinky tail phenotype. Somatic transfer of genomic imprinting between homologs by means of a transvection-like process between paired Tme and T loci is proposed as a model to explain the results obtained.     

Copy Number Variation at the APOL1 Locus

Two coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7–30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Regulatory Mutants at the his1 Locus of Yeast

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1--7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auxotrophic parental alleles. The double mutants, HIS1--7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1--7 background, the mutant regulatory site (HIS1-e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine.