Xp deletions associated with autism in three females

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome. Received: 6 April 1998 / Accepted: 4 November 1998     

Mitochondrial DNA deletions are associated with non-B DNA conformations

Mitochondrial DNA (mtDNA) deletions are a primary cause of mitochondrial disease and are believed to contribute to the aging process and to various neurodegenerative diseases. Despite strong observational and experimental evidence, the molecular basis of the deletion process remains obscure. In this study, we test the hypothesis that the primary cause of mtDNA vulnerability to breakage resides in the formation of non-B DNA conformations, namely hairpin, cruciform and cloverleaf-like elements. Using the largest database of human mtDNA deletions built thus far (753 different cases), we show that site-specific breakage hotspots exist in the mtDNA. Furthermore, we discover that the most frequent deletion breakpoints occur within or near predicted structures, a result that is supported by data from transgenic mice with mitochondrial disease. There is also a significant association between the folding energy of an mtDNA region and the number of breakpoints that it harbours. In particular, two clusters of hairpins (near the D-loop 3'-terminus and the L-strand origin of replication) are hotspots for mtDNA breakage. Consistent with our hypothesis, the highest number of 5'- and 3'-breakpoints per base is found in the highly structured tRNA genes. Overall, the data presented in this study suggest that non-B DNA conformations are a key element of the mtDNA deletion process.     

Replication strand preference for deletions associated with DNA palindromes

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E . coli , in bacteriophage λ and in high-copy-number pUC-based plasmids.     

Activation of expression of a cloned archaebacterial gene in Escherichia coli by IS2, IS5, or deletions

Summary A DNA fragment from the methanogenic archaebacterium Methanococcus voltae, when cloned into the PstI site of the plasmid vector pBR322, complements the Escherichia coli argG mutation strongly or weakly depending on its orientation. Faster-growing variants derived from a strain containing the poorly expressed fragment were found to harbor plasmids which had undergone genetic rearrangements. Some of the plasmids were shown to have acquired an insertion element (IS2 or IS5), derived from the E. coli chromosome, close to the region essential for complementing activity. Other plasmids exhibited no homology with E. coli chromosomal DNA. These were found to represent multimeric forms of the parental plasmid in which 2–3 kb of DNA between the tet promoter and the argG-complementing region had been deleted. Growth rates of the variant strains in the absence of arginine varied significantly, suggesting differences in efficiency of activation of the cloned DNA.     

The sequence of IS4

Summary IS-elements are devoid of easily recognizable transacting functions and exert their visible effects in the position cis only (recent reviews Calos and Miller 1980; Starlinger 1980). It has been a matter of debate, whether these elements encode functions for their own transposition. In the case of the E. coli IS-elements this could not easily be determined by genetic methods, because most of these elements are present in several copies (Saedler and Heiss 1973; Deonier et al. 1979). In the case of the IS-elements flanking transposons, evidence has recently been brought forward that these carry the transposition specificity (Rothstein et al. 1980; Kleckner 1980; Grindley 1981).IS4 is present in one copy only in several E. coli K12 strains and should, therefore, be suitable for genetic and physiological studies (Chadwell et al. 1979). It has been cloned from several sites on the E. coli chromosome in pBR322 (Klaer and Starlinger 1980). Here we report the DNA sequence of IS4 which contains an open reading frame for 442 amino acids, and of the junctions of this element with surrounding DNA at three different sites in E. coli chromosome.     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

Coding region deletions associated with the major form of rDNA interruption in Drosophila

The nucleotide sequences at and around the termini of 5 kb type 1 interruptions in three separate clones of D. melanogaster rDNA repeats have been determined, and have been compared with the sequence of the corresponding region of an insertion-free rDNA repeat. All three interrupted rDNA repeats contain a small deletion of 28S rRNA coding material at the left coding/insertion sequence junction. A second deletion was found in one of the three clones, ad other aberrations were suggested by the results of restriction enzyme digestions of unfractionated rDNA. The termini of 5 kb type 1 rDNA insertions in D. melanogaster were also compared with the corresponding regions of 28S rDNA interruptions in D. virilis: the insertion site is identical in the two species, but the termini of the two species' interruptions show no homology. I sequenced a 1.1 kb region of the 5 kb type 1 D. melanogaster rDNA interruption that covers the sequences of the 1 kb and 0.5 kb insertions. There is 98% homology between the rightmost 1 kb of the 5 kb interruption and the sequences of the shorter insertions. Data suggest that Drosophila rDNA interruptions arose as a transposable element, and that divergence had included length alterations generated by unequal crossing over.     

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.     

Complete maps of IS1, IS2, IS3, IS4, IS5, IS30 and IS150 locations in Escherichia coli K12

Summary In this paper complete distribution maps are presented of the seven IS elements 1, 2, 3, 4, 5, 30 and 150. These maps were obtained during the construction of an almost complete restriction map of the Escherichia coli genome of K12 strain BHB2600. The positions of IS elements were correlated to this map. The distribution of integration sites of all IS types is nonrandom. Besides a large gap from 79 min to 96 min, there is a pronounced IS cluster at 6 min and another at 97 min, map locations that have low gene incidences on the classical map. One cluster coincides with a region of IS induced rearrangements. The IS distribution pattern was compared to patterns of strains W3110 and HB101.     

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.     

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.     

Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis

Bone homeostasis requires a delicate balance between the activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Various molecules coordinate osteoclast function with that of osteoblasts; however, molecules that mediate osteoclast-osteoblast interactions by simultaneous signal transduction in both cell types have not yet been identified. Here we show that osteoclasts express the NFATc1 target gene Efnb2 (encoding ephrinB2), while osteoblasts express the receptor EphB4, along with other ephrin-Eph family members. Using gain- and loss-of-function experiments, we demonstrate that reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade. In addition, forward signaling through EphB4 into osteoblasts enhances osteogenic differentiation, and overexpression of EphB4 in osteoblasts increases bone mass in transgenic mice. These data demonstrate that ephrin-Eph bidirectional signaling links two major molecular mechanisms for cell differentiation--one in osteoclasts and the other in osteoblasts--thereby maintaining bone homeostasis.     

Physical fine mapping of the choroideremia locus using Xq21 deletions associated with complex syndromes

Characterization of several male-viable deletions and duplications with 20 random DNA probes has enabled us to subdivide the Xq21 region into seven discernible intervals. Almost all of the deletions spanning part of Xq21 are associated with choroideremia and mental retardation, with deafness being another common feature. The gene locus for choroideremia was assigned to interval 3 spanning the loci DXS95, DXS165, and DXS233. Genes for X-linked deafness and mental retardation were tentatively assigned to interval 2. Deletions of intervals 4 through 7 were not associated with any clinical abnormality. We have constructed a preliminary long-range restriction map of intervals 2 and 3 using field-inversion gel electrophoresis. The DXS232, DXS121, and DXS233 loci are located on the same SfiI fragment, whereas the DXS165 and DXS95 loci could not be linked to this cluster using SfiI and SalI.     

Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain

Deletions in the mitochondrial DNA (mtDNA) of Balb/c mouse cerebrums, resembling deletions found in elderly humans or in patients with certain disorders, were detected by PCR. Analysis was carried out on mice of various ages and on mice in which the bilateral common carotid arteries had been incompletely ligated to reconstruct cerebral ischemia. A 3,867 bp mtDNA deletion was present only in old or ischemic mouse groups. Among the non-ischemic groups, it was found in 0 of 12 weaning, 0 of 12 young, and four of eight old mice. Among the ischemic groups, it was found in 12 of 17 young and 11 of 11 old mice. Moreover, the percentage of total mtDNA containing deletions was 22% for the old non-ischemic group, 37% for the young ischemic group, and 69% for the old ischemic group. In addition, PCR analysis detected two other deletions of 3,726 bp and 4,236 bp in 4 of the 11 old ischemic cerebrums. The results indicate that mtDNA deletions are associated with aging, that ischemia increases the incidence of mtDNA deletions, and that mtDNA deletions resulting from ischemia are more likely to occur in old mice than in young mice.